WO2007088229A1 - Oncolytic adenoviruses for the treatment of cancer - Google Patents

Oncolytic adenoviruses for the treatment of cancer Download PDF

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Publication number
WO2007088229A1
WO2007088229A1 PCT/ES2007/000050 ES2007000050W WO2007088229A1 WO 2007088229 A1 WO2007088229 A1 WO 2007088229A1 ES 2007000050 W ES2007000050 W ES 2007000050W WO 2007088229 A1 WO2007088229 A1 WO 2007088229A1
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Prior art keywords
promoter
sequence
adenovirus
oncolytic
tumor
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PCT/ES2007/000050
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Spanish (es)
French (fr)
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Ramon Alemany Bonastre
Manel Maria Cascallo Piqueras
Juan José ROJAS EXPÓSITO
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Dnatrix Inc.
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Priority to EP07704759A priority Critical patent/EP1990418B1/en
Priority to AU2007211434A priority patent/AU2007211434A1/en
Priority to JP2008552833A priority patent/JP5075839B2/en
Priority to CA2640528A priority patent/CA2640528C/en
Publication of WO2007088229A1 publication Critical patent/WO2007088229A1/en
Priority to US12/184,881 priority patent/US20090311219A1/en
Priority to US14/327,840 priority patent/US20150071881A1/en
Priority to US15/144,637 priority patent/US10016470B2/en
Priority to US16/028,037 priority patent/US20190183946A1/en

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    • A—HUMAN NECESSITIES
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    • C12N2820/007—Vectors comprising a special origin of replication system tissue or cell-specific

Definitions

  • the field of the invention is related in general terms to the field of tumor biology.
  • the invention relates to selective replication adenoviruses in tumors, called oncolytics, and their use to inhibit cancer.
  • the current cancer treatment is mainly based on chemotherapy, radiotherapy and surgery.
  • Despite a high cure rate for early-stage cancer most advanced cases of cancer are incurable because they cannot be surgically removed or because the radio or chemotherapeutic doses administered are limited by their toxicity in normal cells.
  • biotechnological strategies have been developed that seek to increase the potency and selectivity of cancer treatments.
  • gene therapy and virotherapy use viruses for therapeutic purposes against cancer.
  • the virus is modified to prevent its replication and to serve as a vehicle or vector of therapeutic genetic material.
  • virotherapy uses viruses that selectively replicate and spread in tumor cells 1 .
  • the tumor cell dies from the cytopathic effect caused by the replication of the virus inside it rather than the effect of a therapeutic gene.
  • the preferential replication in a tumor cell is called oncotropism and the tumor analysis is called oncolysis.
  • Viruses that selectively replicate in tumors are called oncolytics.
  • Cancer virotherapy is much earlier than gene therapy.
  • the first observations of cure of tumors with viruses date from the beginning of the last century.
  • 1912 De Pace obtained tumor regressions after inoculating the virus of rabies in cervical carcinomas 2 . Since then many types of viruses have been injected into tumors for treatment 3 .
  • viruses that have a natural oncotropism such as autonomous parvovirus 4 , vesicular stomatitis virus 5 and reovirus 6 .
  • Other viruses can be genetically engineered to selectively replicate in tumors.
  • Herpes Simplex virus has been made oncotropic by deleting the ribonucleotide reductase gene, an enzymatic activity that is available in cells in active proliferation such as tumor cells 7 .
  • adenovirus due to its low pathogenicity and high capacity to infect tumor cells, has been the virus most used in both virotherapy and cancer gene therapy.
  • Ad5 Human adenovirus type 5
  • Ad5 Human adenovirus type 5
  • CAR Coxsackie-Adenovirus Receptor
  • E1A the genes of the early region 1A
  • E1A binds to the Rb cell protein that is forming a complex with the transcription factor E2F. With this, E2F is released to activate the transcription of other viral genes such as E2, E3 and E4 and of the cellular genes that activate the cell cycle.
  • E1 B binds to p53 to activate the cell cycle and prevent apoptosis of the infected cell.
  • E2 encodes for virus replication proteins.
  • E3 proteins that inhibit the antiviral immune response.
  • E4 for viral RNA transport proteins. The expression of these early genes leads to the replication of the viral DNA and once replicated the promoter that regulates the expression of the late or structural genes that form the capsid is activated.
  • oncolytic adenoviruses Two methods have been used to construct oncolytic adenoviruses: the deletion of viral functions that are not necessary in tumor cells and Ia substitution of viral promoters with selective tumor promoters 1 .
  • the gene to be deleted or regulated belongs preferably to the E1 region, and above all it affects E1a because it controls the expression of the other viral genes.
  • deletions of viral functions for example, the E1b-55K protein has been eliminated. This inactive p53 protein to induce into the infected cell the S phase entry of the cell cycle and prevent cell apoptosis.
  • E1 b-55K An adenovirus mutated in E1 b-55K known as Onyx-015 has been used to treat p53-defective tumors although with little clinical success due to its low propagation capacity or oncolytic potency.
  • Another mutation made in the adenoviral genome to achieve selective replication in tumors affects the CR2 domain of E1a.
  • This domain of E1a mediates the binding to proteins of the Retinoblastoma family (Rb).
  • the pRb proteins block the transition from the Go / G1 phase to the S phase of the cell cycle forming a transcription inhibitor complex together with E2F.
  • E2F When E1a binds to pRb, the transcription factor E2F is released from the pRb-E2F complex and E2F acts as a transcriptional activator of the genes responsible for the passage to the S phase and of viral genes such as E2.
  • the release of E2F is thus a key step for the replication of adenovirus.
  • tumor cells the cell cycle is out of control because pRb is absent or inactivated by hyperphosphorylation and E2F is free. In these cells the inactivation of pRB by E1a is no longer necessary. Therefore, an adenovirus with an E1a mutation called Delta-24 that prevents its binding to pRb can normally be propagated in cells with inactive 9R1 pRb.
  • the E1a promoter has been replaced by various promoters such as the promoter of alpha-fetoprotein, prostate specific antigen (PSA), kallikrein, mucin 1 and osteocalcin 11 "15 .
  • PSA prostate specific antigen
  • kallikrein kallikrein
  • mucin 1 mucin 1
  • osteocalcin 11 "15 .
  • the regulation of several viral genes can be carried out with a different promoter for each viral gene such as the E2F1 promoter for E1a and the telomerase promoter for E4. In that case two promoters must be expressed at high levels to allow viral replication such that oncolytic potency can remain reduced in many tumor cells 20.
  • two viral genes can be regulated with the same promoter as for example in the oncolytic adenovirus Onyx411, in which E1a and E4 are regulated by the E2F1 21 promoter.
  • E1a and E4 are regulated by the E2F1 21 promoter.
  • a particularly interesting promoter used in the oncolytic adenovirus design is the E2F1 promoter 20 '21' 29 to 30.
  • This promoter has two E2F binding sites.
  • the family of E2F transcription factors regulates the transcription of the genes that allow the S-phase entry of the cell cycle. These factors behave as activators when they are free and as repressors when they are bound to the retinoblastoma protein pRb 31 .
  • the binding of pRb to E2F is regulated by phosphorylation of pRb so that the phosphorylation of pRb prevents its binding to E2F.
  • the tumors present alterations in the transduction pathways of signal that results in the hyperphosphorylation of pRb and an increase in free E2F.
  • E2F1 gene genes that respond to E2F are expressed as the E2F1 gene.
  • pRb in a normal quiescent cell pRb is not phosphorylated and remains bound to E2F forming a complex that acts as a transcriptional repressor.
  • oncolytic adenoviruses however, the simple regulation of E1a with the E2F1 promoter results in a low level of selective replication in tumors, of the order of 10 times 20.
  • the regulation of other viral genes in addition to E1a is a possible solution to this low selectivity but presents the drawbacks described in the previous paragraph.
  • OAS403 is an oncolytic adenovirus with E1a regulated with the E2F1 and E4 promoter regulated with the telomerase promoter, which also includes a polyadenylation signal to eliminate transcription from the ITR (inverted terminal repeat) and in which it has relocating the packing signal to the right end of the genome to reduce interference with the E1a 20 promoter.
  • ITR inverted terminal repeat
  • OAS403 it has been seen that the packaging signal and sequences adjacent to E4 change position in genome 22 . It has also been described that even minor modifications of the E4 region induce genomic instability with which strategies based on the modification of the E4 region have been abandoned 22 .
  • Another problem encountered with the E2F1 promoter regardless of its selectivity is the lack of potency.
  • an oncolytic adenovirus with E1a regulated with the E2F1 promoter loses lytic capacity with respect to the wild adenovirus as shown in Ryan et al. 20 and in the examples presented in the present invention.
  • the present invention describes the use of appropriate DNA sequences to achieve the proper functioning of a genome promoter of an oncolytic adenovirus. With these sequences an oncolytic adenovirus is designed that presents greater selectivity and antitumor potency.
  • the use of the elements described in the present invention allows to achieve a high tumor selectivity and oncolytic capacity using only a specific tumor promoter.
  • the use of a single promoter reduces the problems of genomic instability associated with the repetition of the same promoter in the adenoviral genome.
  • the regulation only of E1a avoiding Ia regulation of other viral genes allows the correct temporal regulation of adenoviral genes and prevents genomic instability associated with the modification of the E4 region.
  • the present invention relates to an oncolytic adenovirus for the treatment of cancer containing a human DNA sequence isolating a promoter that confers selective expression to an adenoviral gene.
  • the human DNA sequence is a sequence derived from the locus of myotonic dystrophy.
  • an oncolytic adenovirus in which said adenovirus contains a sequence that optimizes the protein translation of an adenoviral gene regulated by a promoter that confers tumor selectivity.
  • said sequence is the kozak sequence.
  • Another object of the invention is an oncolytic adenovirus for the treatment of cancer that contains a human DNA sequence isolating a selective expression promoter that regulates an adenoviral gene and a sequence that optimizes the protein translation of the same adenoviral gene.
  • the human DNA sequence is a sequence derived from the locus of myotonic dystrophy.
  • Another object of the present invention is an adenovirus that contains a human DNA sequence isolating a selective expression promoter that regulates an adenoviral gene and a sequence that optimizes the protein translation of the same adenoviral gene and also presents mutations in one or more genes. of the group E1a, E1b and E4 to achieve selective replication in tumors.
  • the human DNA sequence is a sequence derived from the locus of myotonic dystrophy.
  • Still another object of the present invention is an oncolytic adenovirus that contains a human DNA sequence isolating a selective expression promoter that regulates an adenoviral gene and a sequence that optimizes the protein translation of the same adenoviral gene and modifications in its capsid to increase its infectivity or direct it to a receptor present in a tumor cell.
  • the human DNA sequence is a sequence derived from the locus of myotonic dystrophy.
  • Still another object of the present invention is an oncolytic adenovirus that contains a human DNA sequence isolating a selective expression promoter that regulates an adenoviral gene and a sequence that optimizes the protein translation of the same adenoviral gene and that said adenovirus in turn contains other genes commonly used in the field of cancer gene therapy such as prodrug activators, tumor suppressors or immunostimulators.
  • the human DNA sequence is a sequence derived from the locus of myotonic dystrophy.
  • Still another object of the present invention is an oncolytic adenovirus that contains a human DNA sequence isolating a selective expression promoter that regulates an adenoviral gene and a sequence that optimizes the protein translation of the same adenoviral gene
  • the adenovirus is a derived human adenovirus. of a serotype between 1 and 50.
  • adenovirus is a human adenovirus of serotype 5.
  • the human DNA sequence is a sequence derived from the locus of myotonic dystrophy.
  • Still another object of the present invention is an oncolytic adenovirus that contains a human DNA sequence isolating the promoter of the human E2F1 gene modified by the addition of E2F binding sites to regulate the expression of an adenoviral gene and a sequence that optimizes the translation Protein of the same gene.
  • the human DNA sequence is a sequence derived from the locus of myotonic dystrophy.
  • Another object of the present invention is a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount of an oncolytic adenovirus that contains a human DNA sequence isolating a selective expression promoter that regulates an adenoviral gene and a sequence that optimizes the protein translation of the same adenoviral gene. and one or more pharmaceutically acceptable carriers and excipients.
  • the human DNA sequence is a sequence derived from the locus of myotonic dystrophy.
  • the present invention also aims at the use of an oncolytic adenovirus containing a human DNA sequence isolating a promoter from selective expression that regulates an adenoviral gene and a sequence that optimizes the protein translation of the same adenoviral gene for the preparation of a medicament for the treatment or prevention of cancer or a pre-malignant condition thereof.
  • the human DNA sequence is a sequence derived from the locus of myotonic dystrophy.
  • the adenovirus of the present invention can be optionally combined with other therapeutic modalities against cancer such as chemotherapy or radiotherapy.
  • the present invention describes an oncolytic adenovirus that contains a human DNA sequence, in particular a sequence derived from the locus of myotonic dystrophy, as an isolating sequence of a selective expression promoter that regulates an adenoviral gene and in turn contains a sequence that optimizes The protein translation of the same adenoviral gene, as well as the use of said oncolytic adenovirus for the treatment or prevention of cancer or a pre-malignant condition thereof.
  • the previously described insulators are not of human origin and have not been used in a context of oncolytic adenoviruses.
  • the locus of myotonic dystrophy is located on human chromosome 13 in position 19q13.3.
  • This locus contains two binding sites of the CTCF protein and a variable number according to each individual of CTG repeats that jointly function as a potent insulator of the effect of enhancers or activators on promoters 32 .
  • Prior to this invention its activity has never been analyzed in a viral genome. Its activity in a viral genome is not obvious since its activity has only been demonstrated in the context of the cell chromosome in which the associated histones may have a role in its functioning. Its human origin offers a superior alternative to the use of the chicken HS4 sequence since the transfer of sequences of non-human origin may have biosafety implications.
  • the present invention describes the use of a sequence optimized for protein translation to increase the levels produced of the adenoviral protein regulated under the tumor-specific promoter.
  • the Regulation of the expression of a viral gene with a selective tumor promoter has the disadvantage that the level of expression is usually reduced with respect to the level of expression observed in Ad5. This lower expression entails the lower replicative potency of the oncolytic adenovirus.
  • the insertion of the kozak sequence at the beginning of the translation of the regulated gene with the selective promoter is capable of restoring the expression levels of the regulated gene.
  • the present invention also describes the strategy of increasing the number of E2F binding sites in the sequence of the human E2F1 promoter to better control the expression of E1a in an oncolytic adenovirus.
  • This increase in E2F binding sites produces a greater expression of E1a in tumor cells and a lower expression of E1a in normal cells, which results in an increase in the tumor selectivity of adenoviral replication.
  • the invention is directed towards the need to find better therapies for cancer including, but not limited to, pancreatic, colon and lung cancer.
  • the cancer treatment with the oncolytic adenovirus containing the human DNA sequence and the sequence that optimizes the protein translation can be performed by direct injection into the tumor or by systemic intravenous administration in patients affected by cancer using standard methods in the field of Gene therapy and adenovirus virotherapy.
  • FIGURE 1 Adenovirus structure of selective replication in tumors characterized by containing a sequence of the locus of myotonic dystrophy isolating a selective expression promoter that regulates an adenoviral gene and a sequence that optimizes the protein translation of the same adenoviral gene.
  • Icovir2 contains the locus sequence of the myotonic dystrophy (DM) isolating the human promoter E2F1 that regulates E1a.
  • Icovir ⁇ also contains the kozak sequence at the beginning of translation of E1a to optimize its translation and thus increase the levels of E1a expression in tumor cells.
  • Icov ⁇ r7 also presents two additional E2F binding sites in the E2F1 promoter.
  • FIGURE 2 Scheme of the operation of an oncolytic adenovirus containing the DM sequence of the locus of the myotonic dystrophy isolating the E2F1 promoter that regulates E1a.
  • E1a contains a sequence that optimizes protein translation.
  • the E2F1 promoter can be modified by inserting additional E2F binding sites to increase its selectivity and potency.
  • the pRB-E2F complex acts as a repressor of the E2F1 promoter through the action of histone deacetylases (HDAC) and E1a is not expressed.
  • HDAC histone deacetylases
  • pRB is hyperphosphorylated or absent and E2F is free. In this way it acts as a transcriptional activator of E1a.
  • the kozak sequence in E1a allows a correct level of expression of E1a.
  • the DM insulator prevents interference from the ITR and adenoviral packaging signal in the modified E2F1 promoter
  • FIGURE 3 Demonstration of the effect on the expression of E1a resulting from the insertion of a DM insulating sequence in front of the E2F1 promoter.
  • E2F1 promoter ICOVIR1
  • ICOVIR2 E2F1 promoter
  • Fadu, SCC25 and SKMel-28 the expression of E1a is less than that obtained with adenoviruses where E1a is not regulated by E2F1.
  • E2F1 promoter isolated or not with DM, does not have the power necessary to allow a level of E1a expression in tumor cells comparable to wild adenovirus.
  • the present invention solves this problem with the insertion of the kozak sequence in E1a and the modification of the E2F1 promoter.
  • FIGURE 4 The DM sequence allows to increase the antitumor selectivity of an oncolytic adenovirus with E1a regulated with the E2F1 promoter.
  • the cells were infected with ICOVIR1 and ICOVIR2. Five days post-infection the cells and their culture media were collected and subjected to three freeze-thaw cycles to release the virus. The amount of virus in the cell extract was determined by infection in HEK293 and anti-hexon staining using the 2Hx-2 monoclonal antibody (ATCC) and a secondary Alexa 488 anti-mouse IgG antibody (Molecular Probes, Eugene, OR).
  • ATCC 2Hx-2 monoclonal antibody
  • a secondary Alexa 488 anti-mouse IgG antibody Molecular Probes, Eugene, OR.
  • ICOVI R2 The presence of the DM isolating sequence in ICOVI R2 results in a lower viral replication in normal cells compared to ICOVI R1 which has the non-isolated E2F1 promoter. It is important to note that in the majority of tumor lines its replicative capacity is less than that of AdwtRGD.
  • the present invention describes the method to increase this replicative capacity by means of the insertion of the kozak sequence in E1a and the modification of the E2F1 promoter.
  • FIGURE 5 Effect of the insertion of the kozak sequence to increase the potency of the promoter isolated with DM.
  • ICOVIR5 is distinguished from ICOVIR2 by containing the kozak sequence at the beginning of translation of E1a. The results show that in normal cells ICOVIR5 does not express E1a by presenting the E2F promoter isolated with DM. In tumor cells, the expression level of E1a is higher in ICOVIR5 than in ICOVIR2, which demonstrates the effect of the kozak sequence to increase the potency of the promoter isolated with DM.
  • FIGURE 6 Oncolytic efficacy in vitro of adenoviruses containing E1a regulated with the E2F1 promoter isolated with the DM sequence and the kozak sequence to optimize the translation of E1a.
  • cytolytic capacity in tumor cells of an adenovirus containing the kozak sequence in E1a and an E2F1 promoter isolated by DM was compared with that of a wild virus and a similar oncolytic virus but without the kozak sequence (ICOVIR2).
  • CPE cytopathic effect that the virus induces is measured as a decrease in the amount of protein in an infected cell monolayer (method BCA 33 ).
  • the cells were seeded in 96-well plates at 30,000 cells per well. The next day the cells were infected with serial dilutions virus from a concentration of 1000 plaque forming units per cell.
  • FIGURE 7 Effect of the modification of the E2F1 promoter to increase its potency when it is isolated with the DM sequence.
  • the oncolytic adenovirus ICOVIR7 is distinguished from ICOVIR5 by having the modified E2F1 promoter.
  • the analysis of the expression of E1a in the tumor line 1.36.1.5 of melanoma by western blot is shown.
  • the expression level of E1a is higher in ICOVIR7 than in ICOVIR5 Io which demonstrates the enhancing role of additional E2F binding sites in
  • ICOVIR7 ICOVIR7. Below is the level of viral production of ICOVIR7, ICOVIR5 and ICOVIR2 in the tumor line 1.36.1.5.
  • the AdwtRGD virus in which E1a is not regulated is used as the maximum production control.
  • ICOVIR7 is capable of propagating with the same power as the AdwtRGD control.
  • FIGURE 8 An adenovirus containing E1a regulated with the E2F1 promoter isolated with the DM sequence and the kozak sequence at the start of translation of E1a can be used for the treatment of tumors.
  • the graph shows the evolution of the tumor volume.
  • ICOVIR2 is able to inhibit tumor growth.
  • the photograph shows the presence of virus in the tumor treated with ICOVIR-2 versus the one treated with PBS.
  • the intravenous systemic treatment with ICOVIR5 of mice with SKMel-28 melanoma tumors is shown below.
  • the mean tumor growth of 8-10 tumors / group ⁇ SE is represented.
  • All treatment regimens with ICOVIR-5 demonstrated oncolytic activity resulting in a significantly different tumor growth suppression to the control group (PBS), p ⁇ 0.05 .
  • the photograph shows the presence of virus in the tumor treated
  • FIGURE 9 In vivo demonstration of the reduction of toxicity after intravenously injecting adenoviruses containing E1a regulated with the E2F1 promoter isolated with the DM sequence and the kozak sequence to optimize the translation of E1a.
  • E1a was administered as described in Figure 9.
  • the expression of E1a was evaluated in liver sections by immunohistochemistry (upper panels).
  • E1a is not detected in animals injected with ICOVIR5.
  • the pathological evaluation of liver sections stained with eosin-hematoxylin indicates a normal appearance of the livers of mice injected with ICOVIR5 (lower panels).
  • the present invention describes the use in the treatment of adenovirus cancer containing E1a regulated with the E2F1 promoter isolated with the DM sequence, the kozak sequence to optimize the translation of E1a and the addition of E2F binding sites in the promoter E2F1.
  • the treatment is based on the selective replication of these viruses in tumors that have the altered retinoblastoma pathway.
  • the retinoblastoma pathway is the set of protein interactions that occur from the cell membrane to the nucleus to regulate the level of phosphorylation of the pRb retinoblastoma protein.
  • the cancer is characterized by an alteration of this pathway so that the pRb protein is hyperphosphorylated or lost.
  • This alteration of pRb causes a loss of binding of pRb to the transcription factor E2F and the increase of free E2F in the nucleus of the tumor cells.
  • This transcription factor binds promoters with specific E2F binding sites, such as the E2F1 promoter, to increase its expression.
  • the mechanism of selective replication in adenovirus tumors containing E1a regulated with the E2F1 promoter isolated with the DM sequence, the kozak sequence at the start of translation of E1a and the addition of E2F binding sites in the E2F1 promoter is based in which the presence of free E2F in tumors activates the expression of the E2F1 promoter in this virus and is indicated in Figure 2 of the present invention.
  • the presence of the DM sequence allows the correct activation of the promoter.
  • the presence of the kozak sequence allows the synthesis of an amount of E1a sufficient to maintain the appropriate replicative and lytic capacity of the oncolytic virus.
  • the presence of additional E2F binding sites in the E2F1 promoter allows to increase the level of E1a expression to maintain the appropriate replicative and lytic capacity of the oncolytic virus.
  • the isolating human DM sequence derived from the locus of myotonic dystrophy is represented by SEQ. ID 1 (from position 368 to 1096 of sequence 1).
  • the DM sequence is characterized by containing two binding sites to the CTCF factor and a variable number of repetitions of the CGT sequence that together function as a potent isolator of transcriptional interference 32 .
  • the DM sequence acts to isolate the effect of enhancers located in the adenovirus packaging sequence close to the E1a promoter.
  • the E1a promoter is replaced by a selective tumor promoter such as the E2F1 promoter and, to isolate this promoter from the enhancers present in the adenoviral packing sequence, the DM sequence is inserted between said packing sequence and the E2F1 promoter.
  • the sequence of the E2F1 promoter is shown in SEQ. ID 1 (from position 1283 to position 1564 of sequence 1). This promoter is characterized by presenting two E2F binding sites organized in imperfect palindromes and four Sp1 binding sites 34 .
  • the sequence of the E2F promoter is modified by the insertion of E2F binding sites additional to those already existing in the wild human promoter (from position 1321 to position 1447 of SEQ. ID 3).
  • the translation of mRNA by eukaryotic ribosomes can be optimized if we insert the CCA / GC sequence C in front of the first translated ATG codon 35 .
  • This sequence was identified by Marylin Kozak and has been called kozak. In the present invention this sequence acts by compensating for the little power observed experimentally when a tumor selective promoter such as the E2F1 promoter isolated with the DM sequence is used to control the expression of E1a (position 1558 to 1562 of SEQ. ID 2).
  • the methods of genetically modified adenovirus construction are well established in the field of gene therapy and adenovirus virotherapy 36 "41.
  • the most commonly used method is based on first constructing the desired genetic modification in a plasmid containing the adenoviral region. modify, then perform homologous recombination in bacteria with a plasmid containing the rest of the viral genome 41.
  • the procedure can be as follows:
  • adenoviruses with the expression of a viral gene regulated by selective promoter isolated with the DM sequence and enhanced with the kozak sequence may contain modifications of their capsid to increase their infectivity or target receptors present in Ia tumor cell
  • the adenoviral capsid proteins have been genetically modified to include ligands that increase infectivity or that direct the virus to a receptor in the tumor cell 47 ⁇ 53 . Directing adenovirus to the tumor can also be achieved with bifunctional ligands that bind the virus on the one hand and the tumor receptor on the other 53 "56.
  • the capsid can be covered with polymers such as polyethylene glycol 57.60 .
  • polymers such as polyethylene glycol 57.60 .
  • Another embodiment of the present invention relates to adenoviruses containing E1a regulated with the E2F1 promoter isolated with the DM sequence, the kozak sequence at the start of translation of E1a and the addition of E2F binding sites in the E2F1 promoter and which in turn contain other genes to increase their cytotoxicity on tumor cells such as the thymidine kinase gene, cytosine deaminase, proapoptotic genes, immunostimulators or tumor suppressors.
  • the adenoviruses described in the present invention are propagated following standard methods in the fields of adenovirology and adenoviral vectors 36 '37 .
  • the preferred method of propagation is by infection of a permissive cell line to the replication of adenovirus containing E1a regulated with the E2F1 promoter isolated with the DM sequence, the kozak sequence at the start of translation of E1a and the addition of sites of E2F binding in the E2F1 promoter.
  • the A549 pulmonary adenocarcinoma line is an example of that line. Propagation is carried out, for example, as follows: A549 cells are grown on plastic cell culture plates and infected using 50 viral particles per cell.
  • the cells are collected and stored in tubes. After centrifugation at 1000g for 5 minutes, the cell precipitate freezes and thaws three times to break the cells.
  • the resulting cell extract is centrifuged at 100Og for 5 minutes and the virus supernatant is loaded on top of a cesium chloride gradient and centrifuged for 1 hour at 35.00Og.
  • the virus band in the gradient is reloaded onto another gradient of cesium chloride and centrifuged for 16 hours at 35.00Og.
  • the virus band is collected and dialyzed against PBS-10% glycerol. The dialyzed virus is aliquot and stored at -8O 0 C.
  • Phosphate buffered saline with 10% glycerol is a standard formulation for adenovirus storage.
  • new formulations have been described that improve the stability of virus 61> 62 .
  • the present invention describes the use of adenoviruses containing E1a regulated with the E2F1 promoter isolated with the DM sequence, the kozak sequence at the start of translation of E1a and the addition of E2F binding sites in the E2F1 promoter to treat cancer.
  • the treatment is based on the selective replication of these viruses in cells with an active RB pathway.
  • the protocols for using the viruses described in the present invention in the treatment of cancer follow the same procedures as those used in the fields of adenovirus virotherapy and adenovirus gene therapy.
  • adenoviruses with selective replication of various proposed in the present invention have been used to treat cancer 9-37 - 63-68.
  • the purified adenovirus in any of the formulations described above is added to the culture medium for the infection of the tumor cells.
  • the adenovirus can be administered loco-regionally by injection into the tumor or into a body cavity where the tumor is located, or systemically by injection into the bloodstream.
  • the treatment of tumors with the adenoviruses described object of the present invention can be combined with other therapeutic modalities such as chemotherapy or radiotherapy.
  • An oncolytic adenovirus with E1a regulated with the E2F1 promoter isolated with the DM sequence expresses E1a v selectively replicates in tumor cells.
  • adenovirus with E1a regulated with the E2F1 promoter isolated with the DM sequence was constructed as follows: To generate ICOVIR-1 (Ad-E2F- ⁇ 24RGD), the human E2F1 promoter was obtained by PCR of human peripheral blood mononuclear cells using oligonucleotides that amplify from base pair -218 to +51 of the E2F-1 promoter (position +1 indicates the start of transcription). The oligonucleotides contained targets of Kpn ⁇ and HindW restriction for cloning in the plasmid pGL3 (Promega, South Hampton, UK). The resulting plasmid was named pGL3-E2F.
  • pE2F- ⁇ 24 was obtained by recombination with a pyramid containing 5,766 base pairs from the left end of the adenoviral genome except nucleotides (nt) 122 to 129 of E1a (derived from pXC1- ⁇ 24 with a Hind site ⁇ between nt 348 and nt 522 of the genome of Ad5 9 ).
  • pE2F- ⁇ 24 was recombined with pShuttle 41 to obtain pShuttle-E2F- ⁇ 24.
  • This plasmid was linearized with Pme ⁇ and recombined with pVK503 (which contains the Ad5 sequence with the fiber modified with RGD 69 ) to generate the plasmid pAd-E2F- ⁇ 24RGD or pICOVIR-1.
  • pVK503 which contains the Ad5 sequence with the fiber modified with RGD 69
  • pICOVIR-1 plasmid pAd-E2F- ⁇ 24RGD or pICOVIR-1.
  • the ICOVIR1 virus was generated by digestion with Pac ⁇ of this pyramid and transfection in HEK293 cells.
  • a parallel protocol was used to generate ICOVIR-2 (Ad-DM-E2F- ⁇ 24RGD).
  • the DM-1 isolating sequence was obtained from PCR of human peripheral blood mononuclear cells using oligonucleotides that amplify from nt 13006 to nt 13474 of the DM1 locus (sequence published in GenBank with number L08835).
  • the oligonucleotides of the PCR were designed to incorporate flanking Xho I sites.
  • the DM-1 was subcloned into Xhol of pShuttle-E2F- ⁇ 24 described above to obtain pShuttle-DM-E2F- ⁇ 24.
  • the correct orientation of the DM1 fragment was verified by restriction with ⁇ aA7? H1, HinulW, Xhol and Sma ⁇ .
  • pShuttle-DM-E2F- ⁇ 24 was recombined with pVK503 to generate plCOVIR2.
  • the ICOVIR2 virus was generated by Paci digestion of this pyramid and transfection in HEK293 cells.
  • ICOVIR1 and ICOVIR2 were propagated in the A549 line and purified by methods described in gene therapy and virotherapy 36 .
  • oligonucleotides used for these sequencing are: DMI-Up ( ⁇ '-GGGCAGATGGAGGGCCTTTTATTC-S 1 ), E2F-Up (5'-GTGTTACTCATAGCGCGTAA-3 '), ⁇ 24-down (5'-
  • an oncolytic adenovirus with E1a regulated with the E2F1 promoter isolated with the DM sequence expressly expresses E1a in tumor cells
  • normal cell cultures murine and human hepatocytes, human fibroblasts and human HUVEC endothelial cells
  • tumor cells were infected (NP9 pancreatic carcinoma cells, A549 lung carcinoma, FaDu and SCC25 head and neck carcinomas, and SK-Mel-28 and 1.36.1.5 melanoma) with ICOVIR1 and ICOVIR2 using multiplicity of infection that allowed more than 80% infection .
  • the cells were used in lysis buffer (400 mM NaCI, 1 mM EDTA, 5 mM NaF, 10% glycerol, 1mM sodium orthovanadate, 0.5% Nonidet P-40, 100 ug / ml fluoride phenylmethylsulfonyl, 1 ug / ml leupeptin and 10 ug / ml aprotinin in 1OmM Tris-HCI (pH 7.4)) for 1 h at 4 0 C.
  • lysis buffer 400 mM NaCI, 1 mM EDTA, 5 mM NaF, 10% glycerol, 1mM sodium orthovanadate, 0.5% Nonidet P-40, 100 ug / ml fluoride phenylmethylsulfonyl, 1 ug / ml leupeptin and 10 ug / ml aprotinin in 1OmM Tris-HCI (pH 7.4)
  • the lysate was centrifuged at 1400Og, and the protein supernatant was electrophoresed in 10% SDS -PAGE (25 ug / lane, determined by Bradford, BioRad, CA, USA) and transferred to nitrocellulose (Schleicher and Schuell, Dassel, Germany).
  • the membrane was blocked with 5% skim milk, 0.05% Tween 20, 0.9% NaCI in 5OmM Tris (pH 7.5), and incubated 16h at 4 0 C with a polyclonal anti-adenovirus-2 E1a antibody (clone 13 S- 5, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA).
  • E1a was revealed with a secondary anti-rabbit IgG antibody (DAKO A / S) conjugated to peroxidase and the chemiluminescence protocol of "Amersham's Enhanced Chemioluminescence” (Amersham, Arlington Heights, IL, EUU). The result is shown in Figure 3 of the present invention. It is demonstrated that the presence of the E2F1 promoter (ICOVIR1) is capable of decreasing the expression of E1a in normal cells. However, the DM sequence confers greater control of the expression of E1a by the E2F promoter (ICOVIR2).
  • both ICOVIR1 and ICOVIR2 are capable of expressing E1a, but it is important to note that in some tumor lines such as FaDu, SCC25 and SKMel-28 the expression of E1a is less than that obtained with the wild adenovirus and the oncolytic AdD24RGD where E1a It is not regulated by E2F1. This indicates that the E2F1 promoter isolated or not with DM does not have the power necessary to allow a level of expression of E1 a in tumor cells comparable to wild adenovirus.
  • ICOVIR1 The presence of the E2F1 promoter in ICOVIR1 reduces viral replication in normal cells (fibroblasts and HUVEC). However, the isolating sequence in ICOVIR2 results in a lower viral replication. In certain lines, tumor cells such as A549, ICOVIR1 and ICOVIR2 show a level of replication similar to the Adwt wild adenovirus but in the majority of tumor lines its replicative capacity is lower than that of Adwt.
  • EXAMPLE 2 The kozak sequence allows to increase the expression of E1a an oncolytic adenovirus in which the expression of E1a is regulated with the E2F1 promoter isolated with the DM sequence.
  • An oncolytic adenovirus was constructed with E1a regulated with the E2F1 promoter isolated with the DM sequence and with the kozak sequence to increase its translation.
  • E1a a DNA fragment containing the DM sequence, the E2F1 and E1a promoter was isolated from the pShuttle-DM-E2F-D24 described in example 1 by restriction with Kpn1 and subcloned into pGEM3Z (Promega) obtaining plasmid pGEM-E2F- d24.
  • This plasmid was used to replace the translation start of E1a using oligonucleotides with the kozak sequence obtaining pGEM-E2F-KD24.
  • the Kpn1 fragment thus modified was reclassified in Kpn1 of pShuttle-DM-E2F-D24 to obtain pShuttle- DM-E2F-KD24. Finally pShuttle-DM-E2F-KD24 was recombined with pVK503 to obtain pICOVIR ⁇ .
  • the ICOVIR5 virus was generated by digestion with Pac ⁇ of this plasmid and transfection in HEK293 cells.
  • ICOVIR5 was propagated in the A549 line and purified by methods described in gene therapy and virotherapy 36 . Its structure is presented in Figure 1 of the present invention. The correct sequence of the promoter and E1a was verified by restriction and sequencing. The sequence obtained is shown in SEQ. ID 2.
  • E1a is conditionally expressed in tumor cells when its expression is regulated with the E2F1 promoter isolated with the DM sequence and in addition its translation is optimized with the kozak sequence
  • the expression of E1a was analyzed as described in the Example 1.
  • ICOVIR5 oncolytic adenovirus was included, which is distinguished from ICOVIR2 because it contains the kozak sequence at the beginning of E1a translation.
  • Figure 5 of the present invention In normal cells ICOVIR5 does not express E1a by presenting the E2F promoter isolated with DM. In tumor cells, the expression level of E1a is higher in ICOVIR5 than in ICOVIR2, which demonstrates the effect of the kozak sequence to increase the potency of the promoter isolated with DM.
  • the kozak sequence allows to increase the oncolytic potency of an adenovirus in which the expression of E1a is regulated with the E2F1 promoter isolated with the DM sequence.
  • the cells of the SKMel-28 and FaDu tumor lines were cultured in 96-well plates in which a decrease in the replicative capacity of ICOVIR2 had been seen (as described in Example 1 and Figure 4). These cells were infected with increasing amounts of ICOVIR5, ICOVIR2 and AdwtRGD (the latter used as a control of maximum lithic power). Five days post-infection the amount of protein was spectrophotometrically evaluated as a reflection of cell survival. The results are shown in Figure 6 of the present invention.
  • the lithic capacity of ICOVIR5 in SKMel-28 is the same as that of AdwtRGD and greater than ICOVIR2. In FaDu it is also superior to ICOVIR2 although it does not reach the level of AdwtRGD.
  • E2F1 promoter by insertion of E2F binding sites allows the expression of E1a to be increased in tumor cells when E1a is regulated with the E2F1 promoter isolated with the DM sequence and also its translation is optimized with the kozak sequence.
  • plasmid pGEM-E2FKE1ad24 described in example 2 was introduced by means of directed mutagenesis for BsiWI in the E2F1 promoter (position 1326).
  • BsiWI site two copies of oligonucleotides were ligated with the palindromic sequence of E2F binding and which had ends compatible with BsiWI.
  • the promoter thus modified was sublconized in Kpn1 of pShuttle-DM-E2F-D24 to obtain pShDME2FBsiE2F2KE1ad24.
  • plasmid plCOVIR7 was obtained.
  • the ICOVIR7 virus was generated by Paci digestion of this plasmid and transfection into HEK293 cells.
  • ICOVIR7 was propagated in line A549 and purified by methods described in gene therapy and virotherapy 36 . Its structure is presented in Figure 1 of the present invention. The correct sequence of the promoter and E1a was verified by restriction and sequencing. The sequence obtained is shown in SEQ. ID 3.
  • E1a in the 1.36.1.5 tumor line of melanoma by western blot was analyzed as described in example 1.
  • Oncolytic adenovirus ICOVIR7 is distinguished from ICOVIR5 by having the modified E2F1 promoter. The results are shown in Figure 7 of the present invention.
  • the expression level of E1a is higher in ICOVIR7 which demonstrates the potentiating role of additional E2F binding sites in ICOVIR7.
  • the expression of E1a is higher in ICOVIR5 than in ICOVIR2, Io which demonstrates again the effect of the kozak sequence to increase the potency of the promoter isolated with DM.
  • EXAMPLE 5 An adenovirus containing E1a regulated with the E2F1 promoter isolated with the DM sequence and the kozak sequence at the start of translation of E1a can be used to effectively treat tumors.
  • mice of the Balb / c strain that contained NP9 tumors.
  • Tumors in the control group received two intratumoral injections of saline buffer (2 x 10 ⁇ l).
  • Those in the icovir ⁇ -treated group received two intratumoral injections (2 x 10 ⁇ l) of icovir ⁇ (10 9 viral particles per tumor).
  • V (mm 3 ) A (mm) x B 2 (mm 2 ) x ⁇ / 6, where B is the transverse length.
  • Figure 8 shows the tumor volume with respect to the start of treatment (day 0). The results are presented as mean + SEM The existence of significant differences between the results was calculated using a non-parametric trial of unpaired Mann-Whitney data. The growth curves were compared using an analysis of the variance. The results were considered significant if p ⁇ 0.05. Calculations were performed with the SPSS statistical package (SPSS Inc., Chicago, IL). There is a significant difference between tumor size on days 16 and 21.
  • ICOVIR-5 treatment regimens demonstrated oncolytic activity resulting in a significantly different tumor growth suppression to the control group (PBS), p ⁇ 0.05.
  • the administration of a pre-dose of 3.10 10 vp before the injection of 1.10 11 vp gives this regimen significantly better effectiveness than the other guidelines (p ⁇ 0.05).
  • the different sections of the tumors frozen in OCT were treated with an ⁇ -hexon antibody (adenovirus capsid protein) and were counterstained with 4 ', 6'-diamininid ⁇ 2-phenylndol.
  • the antitumor activity of ICOVIR-5 correlates with the replication of adenovirus at the intratumoral level, evaluated in tumors obtained at day 22 post-injection.
  • the samples of all the groups treated with ICOVI R-5 are positive for the presence of adenovirus, which locates in areas of tumor necrosis.
  • the toxicity associated with the systemic administration of adenovirus is reduced when an adenovirus containing E1a regulated with the E2F1 promoter isolated with the DM sequence and the kozak sequence is used at the beginning of E1a translation.
  • the in vivo toxicity of an adenovirus containing the kozak sequence in E1a and an E2F1 promoter isolated by DM (ICOVIR5) was compared with that of a wild virus and the oncolytic virus AdD24RGD expressing E1a under the wild promoter.
  • the viruses were administered intravenously at different doses and at 5 days post-injection parameters associated with toxicity such as animal survival, body weight, serum transaminase level, and hematogram were evaluated.
  • the lethal dose value 50 (LD 50 ) for AdwtRGD or Ad ⁇ 24RGD in immunocompetent mice Balb / C stands at 5.10 10 viral particles (vp) / mouse at day 5 post-injection, while double this dose (1.10 11 vp / mouse) is only lethal for 10% of mice (LD 1 0) injected with ICOVIR-5.
  • the body weight of the mice injected with 5.10 10 vp of AdwtRGD or Ad ⁇ 24RGD at day 5 post-injection experienced significant losses against the increase in weight of the mice injected with ICOVIR-5.
  • the adenovirus type 5 E1A enhancer contains two functionally distinct domains: one is specific for E1A and the other modulates all early units ⁇ n cis. CeII. 1986; 45: 229-236. Buvoli M, Langer SJ, Bialik S, Leinwand LA. Potential limitations of transcription terminators used as transgene insulators in adenoviral vectors.
  • Suzuki K et al. A conditionally replicative adenovirus with enhanced infectivity shows improved oncolytic potency.
  • Kasono K et al. Selective gene delivery to head and neck cancer cells via an integrin targeted adenoviral vector.
  • Hemminki A et al. Targeting oncolytic adenoviral agents to the epidermal growth factor pathway with a secretory fusion molecule.
  • Gu DL et al. Fibroblast growth factor 2 retargeted adenovirus has redirected cellular tropism: evidence for reduced toxicity and enhanced antitumor activity in mice.

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Abstract

The invention relates to an oncolytic adenovirus for the treatment of cancer, containing a human DNA sequence isolating a promoter conferring selective expression on an adenoviral gene. Said adenovirus can also contain a sequence that optimises the protein translation of an adenoviral gene regulated by a promoter conferring tumour selectivity. The invention is suitable for use in the treatment of cancer.

Description

ADENOVIRUS ONCOLITICOS PARA EL TRATAMIENTO DEL CÁNCER ADENOVIRUS ONCOLITICOS FOR THE TREATMENT OF CANCER
CAMPO DE LA INVENCIÓNFIELD OF THE INVENTION
El campo de Ia invención está relacionado en términos generales con el campo de Ia biología tumoral. En particular Ia invención se refiere a adenovirus de replicación selectiva en tumores, llamados oncolíticos, y su uso para inhibir el cáncer.The field of the invention is related in general terms to the field of tumor biology. In particular, the invention relates to selective replication adenoviruses in tumors, called oncolytics, and their use to inhibit cancer.
ANTECEDENTES DE LA INVENCIÓNBACKGROUND OF THE INVENTION
El tratamiento actual del cáncer se basa principalmente en Ia quimioterapia, radioterapia y cirugía. Pese a una elevada tasa de curación para el cáncer en estadios tempranos, Ia mayoría de casos avanzados de cáncer son incurables porque no pueden ser extirpados quirúrgicamente o porque las dosis radio o quimioterapéuticas administradas se ven limitadas por su toxicidad en células normales. Para paliar esta situación se han desarrollado estrategias biotecnológicas que buscan aumentar Ia potencia y selectividad de los tratamientos oncológicos. Entre ellas, Ia terapia génica y Ia viroterapia utilizan virus con propósitos terapéuticos contra el cáncer. En terapia génica el virus se modifica para impedir su replicación y para servir de vehículo o vector de material genético terapéutico. Por el contrario, Ia viroterapia utiliza virus que se replican y propagan selectivamente en las células tumorales 1. En viroterapia Ia célula tumoral muere por el efecto citopático causado por Ia replicación del virus en su interior más que por el efecto de un gen terapéutico. La replicación preferencial en una célula tumoral se denomina oncotropismo y Ia tisis del tumor se denomina oncolisis. Los virus que se replican selectivamente en tumores se denominan oncolíticos.The current cancer treatment is mainly based on chemotherapy, radiotherapy and surgery. Despite a high cure rate for early-stage cancer, most advanced cases of cancer are incurable because they cannot be surgically removed or because the radio or chemotherapeutic doses administered are limited by their toxicity in normal cells. To alleviate this situation, biotechnological strategies have been developed that seek to increase the potency and selectivity of cancer treatments. Among them, gene therapy and virotherapy use viruses for therapeutic purposes against cancer. In gene therapy the virus is modified to prevent its replication and to serve as a vehicle or vector of therapeutic genetic material. On the contrary, virotherapy uses viruses that selectively replicate and spread in tumor cells 1 . In virotherapy, the tumor cell dies from the cytopathic effect caused by the replication of the virus inside it rather than the effect of a therapeutic gene. The preferential replication in a tumor cell is called oncotropism and the tumor analysis is called oncolysis. Viruses that selectively replicate in tumors are called oncolytics.
La viroterapia del cáncer es muy anterior a Ia terapia génica. Las primeras observaciones de curaciones de tumores con virus datan de principios del siglo pasado. Ya en 1912 De Pace obtuvo regresiones tumorales tras inocular el virus de la rabia en carcinomas cervicales 2. Desde entonces muchos tipos de virus se han inyectado en tumores para su tratamiento 3. Hay virus que presentan un oncotropismo natural como por ejemplo el parvovirus autónomo 4, el virus de Ia estomatitis vesicular 5 y el reovirus 6. Otros virus se pueden manipular genéticamente para que se repliquen selectivamente en tumores. Por ejemplo el Herpes Simplex virus (HSV) se ha hecho oncotrópico al deleccionar el gen de Ia ribonucleótido reductasa, una actividad enzimática dispensable en células en proliferación activa como las células tumorales 7. Sin embargo, el adenovirus, por su baja patogenicidad y alta capacidad de infectar células tumorales ha sido el virus más utilizado tanto en viroterapia como en terapia génica del cáncer.Cancer virotherapy is much earlier than gene therapy. The first observations of cure of tumors with viruses date from the beginning of the last century. Already in 1912 De Pace obtained tumor regressions after inoculating the virus of rabies in cervical carcinomas 2 . Since then many types of viruses have been injected into tumors for treatment 3 . There are viruses that have a natural oncotropism such as autonomous parvovirus 4 , vesicular stomatitis virus 5 and reovirus 6 . Other viruses can be genetically engineered to selectively replicate in tumors. For example, Herpes Simplex virus (HSV) has been made oncotropic by deleting the ribonucleotide reductase gene, an enzymatic activity that is available in cells in active proliferation such as tumor cells 7 . However, adenovirus, due to its low pathogenicity and high capacity to infect tumor cells, has been the virus most used in both virotherapy and cancer gene therapy.
El adenovirus humano tipo 5 (Ad5) es un virus formado por una cápsida proteica icosaédrica que encierra un ADN lineal de 36 kilobases 8. En adultos Ia infección con Ad5 suele ser asintomática y en niños causa un resfriado común y conjuntivitis. En general el Ad5 infecta células epiteliales, que en el curso de una infección natural son las células del epitelio bronquial. Entra en Ia célula por medio de Ia interacción de Ia fibra, proteína viral que se extiende a modo de antena desde los doce vértices de Ia cápsida, con una proteína celular implicada en adhesión intercelular llamada Receptor de Coxsackie-Adenovirus (CAR). Cuando el ADN viral llega al interior del núcleo empieza ordenadamente Ia transcripción de los genes tempranos del virus. Los primeros genes virales que se expresan corresponden a los genes de Ia región temprana 1A (E1A). E1A se une a Ia proteína celular Rb que está formando un complejo con el factor de transcripción E2F. Con ello se libera E2F para activar Ia transcripción de otros genes virales como E2, E3 y E4 y de los genes celulares que activan el ciclo celular. Por otro lado, E1 B se une a p53 para activar el ciclo celular e impedir Ia apoptosis de Ia célula infectada. E2 codifica para proteínas de replicación del virus. E3 para proteínas que inhiben Ia respuesta inmune antiviral. E4 para proteínas de transporte de ARN viral. La expresión de estos genes tempranos conduce a Ia replicación del ADN viral y una vez replicado se activa el promotor que regula Ia expresión de los genes tardíos o estructurales que forman Ia cápsida.Human adenovirus type 5 (Ad5) is a virus formed by an icosahedral protein capsid that encloses a linear DNA of 36 kilobases 8 . In adults, infection with Ad5 is usually asymptomatic and in children it causes a common cold and conjunctivitis. In general, Ad5 infects epithelial cells, which in the course of a natural infection are bronchial epithelial cells. It enters the cell through the interaction of the fiber, a viral protein that extends as an antenna from the twelve vertices of the capsid, with a cellular protein involved in intercellular adhesion called Coxsackie-Adenovirus Receptor (CAR). When the viral DNA reaches the interior of the nucleus, the transcription of the early genes of the virus begins orderly. The first viral genes that are expressed correspond to the genes of the early region 1A (E1A). E1A binds to the Rb cell protein that is forming a complex with the transcription factor E2F. With this, E2F is released to activate the transcription of other viral genes such as E2, E3 and E4 and of the cellular genes that activate the cell cycle. On the other hand, E1 B binds to p53 to activate the cell cycle and prevent apoptosis of the infected cell. E2 encodes for virus replication proteins. E3 for proteins that inhibit the antiviral immune response. E4 for viral RNA transport proteins. The expression of these early genes leads to the replication of the viral DNA and once replicated the promoter that regulates the expression of the late or structural genes that form the capsid is activated.
Se han usado dos métodos para construir adenovirus oncolíticos: Ia delección de funciones virales que no son necesarias en células tumorales y Ia substitución de promotores virales por promotores selectivos de tumor 1. En ambas estrategias el gen a deleccionar o regular pertenece preferiblemente a Ia región E1 , y sobretodo afecta a E1a porque controla Ia expresión de los demás genes virales. En cuanto a delecciones de funciones virales se ha eliminado por ejemplo Ia proteína E1b-55K. Esta proteína inactiva p53 para inducir en Ia célula infectada Ia entrada en fase S del ciclo celular e impedir Ia apoptosis celular. Un adenovirus mutado en E1 b-55K conocido como Onyx-015 se ha usado para tratar tumores defectivos en p53 aunque con escaso éxito clínico por su baja capacidad de propagación o potencia oncolítica. Otra mutación realizada en el genoma adenoviral para conseguir replicación selectiva en tumores afecta al dominio CR2 de E1a. Este dominio de E1a media Ia unión a las proteínas de Ia familia del Retinoblastoma (Rb). Las proteínas pRb bloquean Ia transición de Ia fase Go/G1 a Ia fase S del ciclo celular formando un complejo inhibidor de Ia transcripción junto con E2F. Cuando E1a se une a pRb se libera el factor de transcripción E2F del complejo pRb-E2F y E2F actúa como un activador transcripcional de los genes responsables del paso a Ia fase S y de genes virales como E2. La liberación de E2F es de este modo un paso clave para Ia replicación del adenovirus. En células tumorales el ciclo celular está fuera de control debido a que pRb está ausente o inactivado por hiperfosforilación y E2F está libre. En estas células Ia inactivación de pRB por E1a ya no es necesaria. Por ello un adenovirus con una mutación en E1a llamada Delta-24 que impide su unión a pRb se puede propagar normalmente en células con pRb inactivo 9l1°.Two methods have been used to construct oncolytic adenoviruses: the deletion of viral functions that are not necessary in tumor cells and Ia substitution of viral promoters with selective tumor promoters 1 . In both strategies the gene to be deleted or regulated belongs preferably to the E1 region, and above all it affects E1a because it controls the expression of the other viral genes. As for deletions of viral functions, for example, the E1b-55K protein has been eliminated. This inactive p53 protein to induce into the infected cell the S phase entry of the cell cycle and prevent cell apoptosis. An adenovirus mutated in E1 b-55K known as Onyx-015 has been used to treat p53-defective tumors although with little clinical success due to its low propagation capacity or oncolytic potency. Another mutation made in the adenoviral genome to achieve selective replication in tumors affects the CR2 domain of E1a. This domain of E1a mediates the binding to proteins of the Retinoblastoma family (Rb). The pRb proteins block the transition from the Go / G1 phase to the S phase of the cell cycle forming a transcription inhibitor complex together with E2F. When E1a binds to pRb, the transcription factor E2F is released from the pRb-E2F complex and E2F acts as a transcriptional activator of the genes responsible for the passage to the S phase and of viral genes such as E2. The release of E2F is thus a key step for the replication of adenovirus. In tumor cells the cell cycle is out of control because pRb is absent or inactivated by hyperphosphorylation and E2F is free. In these cells the inactivation of pRB by E1a is no longer necessary. Therefore, an adenovirus with an E1a mutation called Delta-24 that prevents its binding to pRb can normally be propagated in cells with inactive 9R1 pRb.
Con relación a Ia estrategia de substituir promotores virales por promotores selectivos de tumor, el promotor de E1a se ha reemplazado por diversos promotores como el promotor de Ia alfa-fetoproteína, antígeno prostático específico (PSA), kallikreína, mucina 1 y osteocalcina 11"15. Sin embargo se ha identificado un problema importante en el uso de promotores celulares en el contexto viral: Ia existencia de secuencias virales que interfieren con Ia correcta regulación del promotor y que disminuyen su selectividad 16'17. Esta pérdida de selectividad se ha intentado corregir regulando otros genes virales además de E1a, como E1b, E2 y E4 18>19. La regulación de varios genes virales se puede realizar con un promotor distinto para cada gen viral como por ejemplo el promotor de E2F1 para E1a y el promotor de Ia telomerasa para E4. En tal caso los dos promotores deben expresarse a elevados niveles para permitir Ia replicación viral con Io que Ia potencia oncolítica puede quedar disminuida en muchas células tumorales20. Alternativamente dos genes virales se pueden regular con el mismo promotor como por ejemplo en el adenovirus oncolítico Onyx411, en el que E1a y E4 están regulados por el promotor de E2F1 21. Sin embargo, se ha demostrado que Ia duplicación de las secuencias del promotor en el genoma adenoviral produce inestabilidad genómica por recombinación entre estas secuencias repetidas 22. Este problema es de difícil solución puesto que cualquier modificación de Ia región E4 parece inducir Ia inestabilidad genómica del adenovirus oncolítico 22. Además Ia regulación transcripcional de los genes adenovirales está controlada temporalmente de modo que E1a activa Ia expresión de los demás genes virales tempranos. Esta regulación es óptima para el ciclo viral y se pierde si el promotor de otros genes virales distintos a E1a es substituido por promotores específicos de tumor. Por otro lado el problema de Ia interferencia entre secuencias virales y el promotor específico utilizado para controlar Ia replicación adenoviral es especialmente importante cuando se quiere regular Ia transcripción de E1a y de E4 puesto que existen "enhancers" y orígenes de transcripción localizados en las repeticiones terminales y en Ia señal de empaquetamiento del adenovirus 23"25. En el campo de vectores no oncolíticos esta interferencia se ha paliado insertando entre el promotor y estos "enhancers" secuencias aislantes derivadas del locus HS4 del gen de Ia B-globina de pollo 26'27. El mecanismo aislante de HS4 se basa en Ia unión de Ia proteína CTCF que inhibe las interacciones entre factores presentes en el enhancer y el promotor 28. La presente invención describe Ia utilización de una secuencia aislante derivada del genoma humano en el contexto del diseño de adenovirus oncolíticos.Regarding the strategy of replacing viral promoters with selective tumor promoters, the E1a promoter has been replaced by various promoters such as the promoter of alpha-fetoprotein, prostate specific antigen (PSA), kallikrein, mucin 1 and osteocalcin 11 "15 . However we have identified a major problem in the use of cell promoters in the viral context:. the existence of viral sequences that interfere with the proper regulation of the promoter and reduce selectivity 16 '17 this loss of selectivity is attempted to correct regulating other viral genes in addition to E1a, such as E1b, E2 and E4 18> 19. The regulation of several viral genes can be carried out with a different promoter for each viral gene such as the E2F1 promoter for E1a and the telomerase promoter for E4. In that case two promoters must be expressed at high levels to allow viral replication such that oncolytic potency can remain reduced in many tumor cells 20. Alternatively two viral genes can be regulated with the same promoter as for example in the oncolytic adenovirus Onyx411, in which E1a and E4 are regulated by the E2F1 21 promoter. However, it has been shown that duplication of the promoter sequences in the adenoviral genome produces genomic instability by recombination between these repeated sequences 22 . This problem is difficult to solve since any modification of the E4 region seems to induce the genomic instability of the oncolytic adenovirus 22 . In addition, the transcriptional regulation of adenoviral genes is temporarily controlled so that E1a activates the expression of the other early viral genes. This regulation is optimal for the viral cycle and is lost if the promoter of viral genes other than E1a is replaced by tumor-specific promoters. On the other hand, the problem of interference between viral sequences and the specific promoter used to control adenoviral replication is especially important when it is desired to regulate the transcription of E1a and E4 since there are "enhancers" and origins of transcription located in terminal repetitions. and in the 23 "25 adenovirus packaging signal. In the field of non-oncolytic vectors this interference has been alleviated by inserting between the promoter and these" enhancers "isolating sequences derived from the HS4 locus of the chicken B-globin gene 26 ' 27. The isolating mechanism of HS4 is based on the binding of the CTCF protein that inhibits the interactions between factors present in the enhancer and the promoter 28. The present invention describes the use of an isolating sequence derived from the human genome in the context of the design of oncolytic adenovirus.
Un promotor particularmente interesante usado en el diseño de adenovirus oncolíticos es el promotor de E2F1 20'21'29-30. Este promotor presenta dos sitios de unión de E2F. La familia de factores de transcripción E2F regula Ia transcripción de los genes que permiten Ia entrada en fase S del ciclo celular. Estos factores se comportan como activadores cuando están libres y como represores cuando están unidos a Ia proteína del retinoblastoma pRb 31. La unión de pRb a E2F se regula por fosforilación de pRb de modo que Ia fosforilación de pRb impide su unión a E2F. Los tumores presentan alteraciones en las vías de transducción de señal que resultan en Ia hiperfosforilación de pRb y un aumento de E2F libre. Por ello, en los tumores se expresan los genes que responden a E2F como el gen de E2F1. Por el contrario, en una célula normal quiescente pRb no está fosforilado y permanece unido a E2F formando un complejo que actúa de represor transcripcional. En adenovirus oncolíticos, sin embargo, Ia simple regulación de E1a con el promotor de E2F1 resulta en un bajo nivel de replicación selectiva en tumores, del orden de 10 veces 20. La regulación de otros genes virales además de E1a es una posible solución a esta baja selectividad pero presenta los inconvenientes descritos en el párrafo anterior. Por ejemplo OAS403 es un adenovirus oncolítico con E1a regulado con el promotor de E2F1 y E4 regulado con el promotor de Ia telomerasa, que además incluye una señal de poliadenilación para eliminar Ia transcripción desde el ITR (repetición terminal invertida) y en el cual se ha relocalizado Ia señal de empaquetamiento al extremo derecho del genoma para disminuir Ia interferencia con el promotor de E1a 20. Durante Ia amplificación de OAS403 se ha visto que Ia señal de empaquetamiento y secuencias adyacentes a E4 cambian de posición en el genoma 22. Se ha descrito además que modificaciones incluso menores de Ia región E4 inducen inestabilidad genómica con Io que se han abandonado las estrategias basadas en Ia modificación de Ia región E4 22. Otro problema encontrado con el promotor de E2F1 independientemente de su selectividad es Ia falta de potencia. Además de ser poco selectivo, un adenovirus oncolítico con E1a regulado con el promotor de E2F1 pierde capacidad lítica respecto al adenovirus salvaje tal como se muestra en Ryan y colaboradores 20 y en los ejemplos presentados en Ia presente invención. La presente invención describe Ia utilización de secuencias de DNA apropiadas para conseguir el correcto funcionamiento de un promotor en genoma de un adenovirus oncolítico. Con estas secuencias se diseña un adenovirus oncolítico que presenta mayor selectividad y potencia antitumoral. La utilización de los elementos descritos en Ia presente invención permite conseguir una elevada selectividad tumoral y capacidad oncolítica utilizando tan solo un promotor específico tumoral. La utilización de un solo promotor disminuye los problemas de inestabilidad genómica asociada a Ia repetición del mismo promotor en el genoma adenoviral. Además, Ia regulación solamente de E1a evitando Ia regulación de otros genes virales, permite Ia correcta regulación temporal de los genes adenovirales y evita Ia inestabilidad genómica asociada a Ia modificación de Ia región E4.A particularly interesting promoter used in the oncolytic adenovirus design is the E2F1 promoter 20 '21' 29 to 30. This promoter has two E2F binding sites. The family of E2F transcription factors regulates the transcription of the genes that allow the S-phase entry of the cell cycle. These factors behave as activators when they are free and as repressors when they are bound to the retinoblastoma protein pRb 31 . The binding of pRb to E2F is regulated by phosphorylation of pRb so that the phosphorylation of pRb prevents its binding to E2F. The tumors present alterations in the transduction pathways of signal that results in the hyperphosphorylation of pRb and an increase in free E2F. Therefore, in tumors, genes that respond to E2F are expressed as the E2F1 gene. On the contrary, in a normal quiescent cell pRb is not phosphorylated and remains bound to E2F forming a complex that acts as a transcriptional repressor. In oncolytic adenoviruses, however, the simple regulation of E1a with the E2F1 promoter results in a low level of selective replication in tumors, of the order of 10 times 20. The regulation of other viral genes in addition to E1a is a possible solution to this low selectivity but presents the drawbacks described in the previous paragraph. For example, OAS403 is an oncolytic adenovirus with E1a regulated with the E2F1 and E4 promoter regulated with the telomerase promoter, which also includes a polyadenylation signal to eliminate transcription from the ITR (inverted terminal repeat) and in which it has relocating the packing signal to the right end of the genome to reduce interference with the E1a 20 promoter. During the amplification of OAS403 it has been seen that the packaging signal and sequences adjacent to E4 change position in genome 22 . It has also been described that even minor modifications of the E4 region induce genomic instability with which strategies based on the modification of the E4 region have been abandoned 22 . Another problem encountered with the E2F1 promoter regardless of its selectivity is the lack of potency. In addition to being unselective, an oncolytic adenovirus with E1a regulated with the E2F1 promoter loses lytic capacity with respect to the wild adenovirus as shown in Ryan et al. 20 and in the examples presented in the present invention. The present invention describes the use of appropriate DNA sequences to achieve the proper functioning of a genome promoter of an oncolytic adenovirus. With these sequences an oncolytic adenovirus is designed that presents greater selectivity and antitumor potency. The use of the elements described in the present invention allows to achieve a high tumor selectivity and oncolytic capacity using only a specific tumor promoter. The use of a single promoter reduces the problems of genomic instability associated with the repetition of the same promoter in the adenoviral genome. In addition, the regulation only of E1a avoiding Ia regulation of other viral genes, allows the correct temporal regulation of adenoviral genes and prevents genomic instability associated with the modification of the E4 region.
DESCRIPCIÓN DE LA INVENCIÓNDESCRIPTION OF THE INVENTION
La presente invención se refiere a un adenovirus oncolítico para el tratamiento del cáncer que contiene una secuencia de DNA humano aislando a un promotor que confiere expresión selectiva a un gen adenoviral. En particular, Ia secuencia de DNA humano es una secuencia derivada del locus de Ia distrofia miotónica.The present invention relates to an oncolytic adenovirus for the treatment of cancer containing a human DNA sequence isolating a promoter that confers selective expression to an adenoviral gene. In particular, the human DNA sequence is a sequence derived from the locus of myotonic dystrophy.
También se refiere a un adenovirus oncolítico en Ia que dicho adenovirus contiene una secuencia que optimiza Ia traducción proteica de un gen adenoviral regulado por un promotor que confiere selectividad tumoral. En particular, dicha secuencia es Ia secuencia de kozak. Otro objeto de Ia invención es un adenovirus oncolítico para el tratamiento del cáncer que contiene una secuencia de DNA humano aislando a un promotor de expresión selectiva que regula un gen adenoviral y una secuencia que optimiza Ia traducción proteica del mismo gen adenoviral. En particular, Ia secuencia de DNA humano es una secuencia derivada del locus de Ia distrofia miotónica. Otro objeto de Ia presente invención es un adenovirus que contiene una secuencia de DNA humano aislando a un promotor de expresión selectiva que regula un gen adenoviral y una secuencia que optimiza Ia traducción proteica del mismo gen adenoviral y que también presenta mutaciones en uno o más genes del grupo E1a, E1b y E4 para conseguir replicación selectiva en tumores. En particular, Ia secuencia de DNA humano es una secuencia derivada del locus de Ia distrofia miotónica.It also refers to an oncolytic adenovirus in which said adenovirus contains a sequence that optimizes the protein translation of an adenoviral gene regulated by a promoter that confers tumor selectivity. In particular, said sequence is the kozak sequence. Another object of the invention is an oncolytic adenovirus for the treatment of cancer that contains a human DNA sequence isolating a selective expression promoter that regulates an adenoviral gene and a sequence that optimizes the protein translation of the same adenoviral gene. In particular, the human DNA sequence is a sequence derived from the locus of myotonic dystrophy. Another object of the present invention is an adenovirus that contains a human DNA sequence isolating a selective expression promoter that regulates an adenoviral gene and a sequence that optimizes the protein translation of the same adenoviral gene and also presents mutations in one or more genes. of the group E1a, E1b and E4 to achieve selective replication in tumors. In particular, the human DNA sequence is a sequence derived from the locus of myotonic dystrophy.
Aún otro objeto de Ia presente invención es un adenovirus oncolítico que contiene una secuencia de DNA humano aislando a un promotor de expresión selectiva que regula un gen adenoviral y una secuencia que optimiza Ia traducción proteica del mismo gen adenoviral y modificaciones en su cápsida para aumentar su infectividad o dirigirlo a un receptor presente en una célula tumoral. En particular, la secuencia de DNA humano es una secuencia derivada del locus de Ia distrofia miotónica.Still another object of the present invention is an oncolytic adenovirus that contains a human DNA sequence isolating a selective expression promoter that regulates an adenoviral gene and a sequence that optimizes the protein translation of the same adenoviral gene and modifications in its capsid to increase its infectivity or direct it to a receptor present in a tumor cell. In In particular, the human DNA sequence is a sequence derived from the locus of myotonic dystrophy.
Aún otro objeto de Ia presente invención es un adenovirus oncolítico que contiene una secuencia de DNA humano aislando a un promotor de expresión selectiva que regula un gen adenoviral y una secuencia que optimiza Ia traducción proteica del mismo gen adenoviral y que dicho adenovirus a su vez contiene otros genes usados comúnmente en el campo de terapia génica del cáncer como activadores de prodrogas, supresores tumorales o inmunoestimuladores. En particular, Ia secuencia de DNA humano es una secuencia derivada del locus de Ia distrofia miotónica.Still another object of the present invention is an oncolytic adenovirus that contains a human DNA sequence isolating a selective expression promoter that regulates an adenoviral gene and a sequence that optimizes the protein translation of the same adenoviral gene and that said adenovirus in turn contains other genes commonly used in the field of cancer gene therapy such as prodrug activators, tumor suppressors or immunostimulators. In particular, the human DNA sequence is a sequence derived from the locus of myotonic dystrophy.
Aún otro objeto de Ia presente invención es un adenovirus oncolítico que contiene una secuencia de DNA humano aislando a un promotor de expresión selectiva que regula un gen adenoviral y una secuencia que optimiza Ia traducción proteica del mismo gen adenoviral donde el adenovirus es un adenovirus humano derivado de un serotipo entre el 1 al 50. En particular, el adenovirus es un adenovirus humano del serotipo 5. En particular, Ia secuencia de DNA humano es una secuencia derivada del locus de Ia distrofia miotónica.Still another object of the present invention is an oncolytic adenovirus that contains a human DNA sequence isolating a selective expression promoter that regulates an adenoviral gene and a sequence that optimizes the protein translation of the same adenoviral gene where the adenovirus is a derived human adenovirus. of a serotype between 1 and 50. In particular, adenovirus is a human adenovirus of serotype 5. In particular, the human DNA sequence is a sequence derived from the locus of myotonic dystrophy.
Aún otro objeto de Ia presente invención es un adenovirus oncolítico que contiene una secuencia de DNA humano aislando al promotor del gen E2F1 humano modificado por Ia adición de sitios de unión a E2F para regular Ia expresión de un gen adenoviral y una secuencia que optimiza Ia traducción proteica del mismo gen. En particular, Ia secuencia de DNA humano es una secuencia derivada del locus de Ia distrofia miotónica.Still another object of the present invention is an oncolytic adenovirus that contains a human DNA sequence isolating the promoter of the human E2F1 gene modified by the addition of E2F binding sites to regulate the expression of an adenoviral gene and a sequence that optimizes the translation Protein of the same gene. In particular, the human DNA sequence is a sequence derived from the locus of myotonic dystrophy.
Otro objeto de Ia presente invención es una composición farmacéutica que comprende una cantidad efectiva de un adenovirus oncolítico que contiene una secuencia de DNA humano aislando a un promotor de expresión selectiva que regula un gen adenoviral y una secuencia que optimiza Ia traducción proteica del mismo gen adenoviral y uno o más portadores y excipientes farmacéuticamente aceptables. En particular, Ia secuencia de DNA humano es una secuencia derivada del locus de Ia distrofia miotónica.Another object of the present invention is a pharmaceutical composition comprising an effective amount of an oncolytic adenovirus that contains a human DNA sequence isolating a selective expression promoter that regulates an adenoviral gene and a sequence that optimizes the protein translation of the same adenoviral gene. and one or more pharmaceutically acceptable carriers and excipients. In particular, the human DNA sequence is a sequence derived from the locus of myotonic dystrophy.
La presente invención también tiene por objeto el uso de un adenovirus oncolítico que contiene una secuencia de DNA humano aislando a un promotor de expresión selectiva que regula un gen adenoviral y una secuencia que optimiza Ia traducción proteica del mismo gen adenoviral para Ia preparación de un medicamento para el tratamiento o prevención del cáncer o de una condición pre- maligna del mismo. En particular, Ia secuencia de DNA humano es una secuencia derivada del locus de Ia distrofia miotónica.The present invention also aims at the use of an oncolytic adenovirus containing a human DNA sequence isolating a promoter from selective expression that regulates an adenoviral gene and a sequence that optimizes the protein translation of the same adenoviral gene for the preparation of a medicament for the treatment or prevention of cancer or a pre-malignant condition thereof. In particular, the human DNA sequence is a sequence derived from the locus of myotonic dystrophy.
El adenovirus de Ia presente invención puede combinarse optativamente con otras modalidades terapéuticas contra el cáncer como Ia quimioterapia o Ia radioterapia.The adenovirus of the present invention can be optionally combined with other therapeutic modalities against cancer such as chemotherapy or radiotherapy.
La presente invención describe un adenovirus oncolítico que contiene una secuencia de DNA humano, en particular una secuencia derivada del locus de Ia distrofia miotónica, como secuencia aislante de un promotor de expresión selectiva que regula a un gen adenoviral y a su vez contiene una secuencia que optimiza Ia traducción proteica del mismo gen adenoviral, así como el uso de dicho adenovirus oncolítico para el tratamiento o prevención del cáncer o de una condición pre-maligna del mismo. Previamente se ha descrito el uso de secuencias aislantes derivadas de Ia B-globina de pollo en vectores adenovirales 26'27. A diferencia de Ia presente invención los aislantes descritos previamente no son de origen humano y no se han utilizado en un contexto de adenovirus oncolíticos. El locus de Ia distrofia miotónica se localiza en el cromosoma 13 humano en Ia posición 19q13.3. Este locus contiene dos sitios de unión de Ia proteína CTCF y un número variable según cada individuo de repeticiones CTG que conjuntamente funcionan como un potente aislante del efecto de los "enhancers" o activadores sobre los promotores32. Previamente a esta invención su actividad nunca ha sido analizada en un genoma viral. Su actividad en un genoma viral no es obvia puesto que sólo se ha demostrado su actividad en el contexto del cromosoma celular en el que las histonas asociadas pueden tener un papel en su funcionamiento. Su origen humano ofrece una alternativa superior al uso de Ia secuencia HS4 de pollo puesto que Ia transferencia de secuencias de origen no humano puede tener implicaciones de bioseguridad. Además Ia presente invención describe Ia utilización de una secuencia optimizada para Ia traducción proteica para aumentar los niveles producidos de Ia proteína adenoviral regulada bajo el promotor específico de tumores. La regulación de Ia expresión de un gen viral con un promotor selectivo tumoral presenta el inconveniente de que el nivel de expresión suele resultar reducido con respecto al nivel de expresión observado en Ad5. Esta menor expresión conlleva Ia menor potencia replicativa del adenovirus oncolítico. La inserción de Ia secuencia de kozak en el inicio de Ia traducción del gen regulado con el promotor selectivo es capaz de restaurar los niveles de expresión del gen regulado.The present invention describes an oncolytic adenovirus that contains a human DNA sequence, in particular a sequence derived from the locus of myotonic dystrophy, as an isolating sequence of a selective expression promoter that regulates an adenoviral gene and in turn contains a sequence that optimizes The protein translation of the same adenoviral gene, as well as the use of said oncolytic adenovirus for the treatment or prevention of cancer or a pre-malignant condition thereof. Previously it described using isolating sequences derived from B-globin of chickens in adenoviral vectors 26 '27. Unlike the present invention, the previously described insulators are not of human origin and have not been used in a context of oncolytic adenoviruses. The locus of myotonic dystrophy is located on human chromosome 13 in position 19q13.3. This locus contains two binding sites of the CTCF protein and a variable number according to each individual of CTG repeats that jointly function as a potent insulator of the effect of enhancers or activators on promoters 32 . Prior to this invention its activity has never been analyzed in a viral genome. Its activity in a viral genome is not obvious since its activity has only been demonstrated in the context of the cell chromosome in which the associated histones may have a role in its functioning. Its human origin offers a superior alternative to the use of the chicken HS4 sequence since the transfer of sequences of non-human origin may have biosafety implications. Furthermore, the present invention describes the use of a sequence optimized for protein translation to increase the levels produced of the adenoviral protein regulated under the tumor-specific promoter. The Regulation of the expression of a viral gene with a selective tumor promoter has the disadvantage that the level of expression is usually reduced with respect to the level of expression observed in Ad5. This lower expression entails the lower replicative potency of the oncolytic adenovirus. The insertion of the kozak sequence at the beginning of the translation of the regulated gene with the selective promoter is capable of restoring the expression levels of the regulated gene.
La presente invención también describe Ia estrategia de aumentar el número de sitios de unión a E2F en Ia secuencia del promotor humano de E2F1 para controlar mejor Ia expresión de E1a en un adenovirus oncolítico. Este aumento de sitios de unión a E2F produce una mayor expresión de E1a en células tumorales y una menor expresión de E1a en células normales, Io que resulta en un aumento de Ia selectividad tumoral de Ia replicación adenoviral.The present invention also describes the strategy of increasing the number of E2F binding sites in the sequence of the human E2F1 promoter to better control the expression of E1a in an oncolytic adenovirus. This increase in E2F binding sites produces a greater expression of E1a in tumor cells and a lower expression of E1a in normal cells, which results in an increase in the tumor selectivity of adenoviral replication.
La invención se dirige hacia Ia necesidad de hallar mejores terapias para el cáncer incluyendo, pero no limitándose a, cáncer de páncreas, colon y pulmón. El tratamiento del cáncer con el adenovirus oncolítico que contiene Ia secuencia de DNA humano y Ia secuencia que optimiza Ia traducción proteica se puede realizar por inyección directa del dentro del tumor o por administración endovenosa sistémica en pacientes afectados de cáncer usando métodos estándar en el campo de terapia génica y viroterapia con adenovirus.The invention is directed towards the need to find better therapies for cancer including, but not limited to, pancreatic, colon and lung cancer. The cancer treatment with the oncolytic adenovirus containing the human DNA sequence and the sequence that optimizes the protein translation can be performed by direct injection into the tumor or by systemic intravenous administration in patients affected by cancer using standard methods in the field of Gene therapy and adenovirus virotherapy.
DESCRIPCIÓN DE LAS FIGURASDESCRIPTION OF THE FIGURES
Los dibujos incluidos en Ia invención se han anexado con el propósito de que las características, ventajas, y construcciones de Ia invención queden claras y se entiendan con detalle. Esos dibujos forman parte de las especificaciones e ilustran las invenciones preferidas pero no deben ser considerados limitativos del ámbito de Ia invención.The drawings included in the invention have been annexed so that the features, advantages, and constructions of the invention are clear and understood in detail. These drawings are part of the specifications and illustrate the preferred inventions but should not be considered as limiting the scope of the invention.
FIGURA 1. Estructura de adenovirus de replicación selectiva en tumores caracterizados por contener una secuencia del locus de Ia distrofia miotónica aislando a un promotor de expresión selectiva que regula un gen adenoviral y una secuencia que optimiza Ia traducción proteica del mismo gen adenoviral. Icovir2 contiene Ia secuencia del locus de Ia distrofia miotónica (DM) aislando al promotor humano E2F1 que regula E1a. Icovirδ contiene además Ia secuencia de kozak en el inicio de traducción de E1a para optimizar su traducción y aumentar así los niveles de expresión de E1a en células tumorales. Icov¡r7 presenta además dos sitios adicionales de unión a E2F en el promotor de E2F1.FIGURE 1. Adenovirus structure of selective replication in tumors characterized by containing a sequence of the locus of myotonic dystrophy isolating a selective expression promoter that regulates an adenoviral gene and a sequence that optimizes the protein translation of the same adenoviral gene. Icovir2 contains the locus sequence of the myotonic dystrophy (DM) isolating the human promoter E2F1 that regulates E1a. Icovirδ also contains the kozak sequence at the beginning of translation of E1a to optimize its translation and thus increase the levels of E1a expression in tumor cells. Icov¡r7 also presents two additional E2F binding sites in the E2F1 promoter.
FIGURA 2. Esquema del funcionamiento de un adenovirus oncolítico que contiene Ia secuencia DM del locus de Ia distrofia miotónica aislando al promotor E2F1 que regula E1a. E1a contiene una secuencia que optimiza Ia traducción proteica. El promotor de E2F1 puede estar modificado mediante Ia inserción de sitios adicionales de unión a E2F para aumentar su selectividad y potencia. En una célula normal el complejo pRB-E2F actúa de represor del promotor de E2F1 a través de Ia acción de deacetilasas de histonas (HDAC) y no se expresa E1a. En una célula tumoral pRB se halla hiperfosforilado o ausente y E2F se halla libre. De esta forma actúa como activador transcripcional de E1a. La secuencia kozak en E1a permite un nivel correcto de expresión de E1a. El aislante DM evita las interferencias del ITR y señal de empaquetamiento adenoviral en el promotor E2F1 modificado.FIGURE 2. Scheme of the operation of an oncolytic adenovirus containing the DM sequence of the locus of the myotonic dystrophy isolating the E2F1 promoter that regulates E1a. E1a contains a sequence that optimizes protein translation. The E2F1 promoter can be modified by inserting additional E2F binding sites to increase its selectivity and potency. In a normal cell, the pRB-E2F complex acts as a repressor of the E2F1 promoter through the action of histone deacetylases (HDAC) and E1a is not expressed. In a tumor cell pRB is hyperphosphorylated or absent and E2F is free. In this way it acts as a transcriptional activator of E1a. The kozak sequence in E1a allows a correct level of expression of E1a. The DM insulator prevents interference from the ITR and adenoviral packaging signal in the modified E2F1 promoter.
FIGURA 3 Demostración del efecto sobre Ia expresión de E1a resultante de Ia inserción Ia una secuencia aislante DM delante del promotor E2F1.FIGURE 3 Demonstration of the effect on the expression of E1a resulting from the insertion of a DM insulating sequence in front of the E2F1 promoter.
Cada tipo celular indicado se infectó con el virus indicado. Después de 24 horas se usaron las células y se detectó E1a por western blot. La presencia del promotor E2F1 (ICOVIR1 ) es capaz de disminuir Ia expresión de EIa en células normales. Sin embargo, se observa que Ia secuencia DM confiere un mayor control de Ia expresión de E1a por el promotor E2F (ICOVIR2). En células tumorales tanto ICOVIR1 como ICOVIR2 son capaces de expresar E1a, pero en Fadu, SCC25 y SKMel-28 Ia expresión de E1a es menor que Ia obtenida con los adenovirus en donde E1a no está regulada por E2F1. Ello indica que el promotor de E2F1 , aislado o no con DM, no tiene Ia potencia necesaria para permitir un nivel de expresión de E1a en células tumorales comparable al adenovirus salvaje. La presente invención resuelve este problema con Ia inserción de Ia secuencia de kozak en E1a y Ia modificación del promotor E2F1.Each indicated cell type was infected with the indicated virus. After 24 hours the cells were used and E1a was detected by western blot. The presence of the E2F1 promoter (ICOVIR1) is capable of decreasing the expression of EIa in normal cells. However, it is observed that the DM sequence confers greater control of the expression of E1a by the E2F promoter (ICOVIR2). In tumor cells, both ICOVIR1 and ICOVIR2 are capable of expressing E1a, but in Fadu, SCC25 and SKMel-28 the expression of E1a is less than that obtained with adenoviruses where E1a is not regulated by E2F1. This indicates that the E2F1 promoter, isolated or not with DM, does not have the power necessary to allow a level of E1a expression in tumor cells comparable to wild adenovirus. The present invention solves this problem with the insertion of the kozak sequence in E1a and the modification of the E2F1 promoter.
FIGURA 4. La secuencia DM permite aumentar Ia selectividad antitumoral de un adenovirus oncolítico con E1a regulada con el promotor de E2F1.FIGURE 4. The DM sequence allows to increase the antitumor selectivity of an oncolytic adenovirus with E1a regulated with the E2F1 promoter.
Para demostrar que un adenovirus oncolítico con E1a regulada con el promotor de E2F1 aislado con Ia secuencia DM se replica selectivamente en células tumorales se procedió a infectar las células con ICOVIR1 e ICOVIR2. Cinco días post-infección se recolectaron las células y sus medios de cultivo y se sometieron a tres ciclos de congelación-descongelación para liberar el virus. La cantidad de virus en el extracto celular se determinó por infección en HEK293 y tinción anti-hexón utilizando el anticuerpo monoclonal 2Hx-2 (ATCC) y un anticuerpo secundario Alexa 488 anti-lgG de ratón (Molecular Probes, Eugene, OR). La presencia de Ia secuencia aislante DM en ICOVI R2 resulta en una menor replicación viral en células normales comparada con ICOVI R1 que tiene el promotor E2F1 no aislado. Es importante señalar que en Ia mayoría de líneas tumorales su capacidad replicativa es menor que Ia de AdwtRGD. La presente invención describe el método para aumentar esta capacidad replicativa mediante Ia inserción de Ia secuencia de kozak en E1a y Ia modificación del promotor E2F1.To demonstrate that an oncolytic adenovirus with E1a regulated with the E2F1 promoter isolated with the DM sequence is selectively replicated in tumor cells, the cells were infected with ICOVIR1 and ICOVIR2. Five days post-infection the cells and their culture media were collected and subjected to three freeze-thaw cycles to release the virus. The amount of virus in the cell extract was determined by infection in HEK293 and anti-hexon staining using the 2Hx-2 monoclonal antibody (ATCC) and a secondary Alexa 488 anti-mouse IgG antibody (Molecular Probes, Eugene, OR). The presence of the DM isolating sequence in ICOVI R2 results in a lower viral replication in normal cells compared to ICOVI R1 which has the non-isolated E2F1 promoter. It is important to note that in the majority of tumor lines its replicative capacity is less than that of AdwtRGD. The present invention describes the method to increase this replicative capacity by means of the insertion of the kozak sequence in E1a and the modification of the E2F1 promoter.
FIGURA 5 Efecto de Ia inserción de Ia secuencia de kozak para aumentar Ia potencia del promotor aislado con DM.FIGURE 5 Effect of the insertion of the kozak sequence to increase the potency of the promoter isolated with DM.
Cada tipo celular indicado a Ia izquierda de Ia figura se infectó con el virus indicado arriba de Ia figura. Después de 24 horas se usaron las células y se detectó E1a por western blot. ICOVIR5 se distingue de ICOVIR2 por contener Ia secuencia de kozak en el inicio de traducción de E1a. Los resultados muestran que en células normales ICOVIR5 no expresa E1a por presentar el promotor E2F aislado con DM. En células tumorales el nivel de expresión de E1a es superior en ICOVIR5 que en ICOVIR2, Io que demuestra el efecto de Ia secuencia de kozak para aumentar Ia potencia del promotor aislado con DM. FIGURA 6. Eficacia oncolítica in vitro de los adenovirus que contienen E1a regulado con el promotor de E2F1 aislado con Ia secuencia DM y Ia secuencia de kozak para optimizar Ia traducción de E1a.Each cell type indicated to the left of the figure was infected with the virus indicated above the figure. After 24 hours the cells were used and E1a was detected by western blot. ICOVIR5 is distinguished from ICOVIR2 by containing the kozak sequence at the beginning of translation of E1a. The results show that in normal cells ICOVIR5 does not express E1a by presenting the E2F promoter isolated with DM. In tumor cells, the expression level of E1a is higher in ICOVIR5 than in ICOVIR2, which demonstrates the effect of the kozak sequence to increase the potency of the promoter isolated with DM. FIGURE 6. Oncolytic efficacy in vitro of adenoviruses containing E1a regulated with the E2F1 promoter isolated with the DM sequence and the kozak sequence to optimize the translation of E1a.
La capacidad citolítica en células tumorales de un adenovirus que contiene Ia secuencia de kozak en E1a y un promotor E2F1 aislado por DM (ICOVIR5) se comparó con Ia de un virus salvaje y un virus oncolítico similar pero sin Ia secuencia de kozak (ICOVIR2) El efecto citopático (CPE) que el virus induce se mide como una disminución de Ia cantidad de proteína en una monocapa celular infectada (método BCA 33). Las células se sembraron en placas de 96 pocilios a 30.000 células por pocilio. Al día siguiente las células se infectaron con diluciones seriadas virus desde una concentración de 1000 unidades formadoras de placa por célula. Las células infectadas se incubaron durante 5 días, se lavaron con PBS y se medió Ia cantidad de proteína restante en el pocilio. En conjunto, los resultados muestran que ICOVIR5 tiene mayor capacidad lítica que ICOVIR2 Io que demuestra el efecto potenciador conferido por Ia secuencia de kozak.The cytolytic capacity in tumor cells of an adenovirus containing the kozak sequence in E1a and an E2F1 promoter isolated by DM (ICOVIR5) was compared with that of a wild virus and a similar oncolytic virus but without the kozak sequence (ICOVIR2). cytopathic effect (CPE) that the virus induces is measured as a decrease in the amount of protein in an infected cell monolayer (method BCA 33 ). The cells were seeded in 96-well plates at 30,000 cells per well. The next day the cells were infected with serial dilutions virus from a concentration of 1000 plaque forming units per cell. Infected cells were incubated for 5 days, washed with PBS and the amount of protein remaining in the well was measured. Together, the results show that ICOVIR5 has a greater lithic capacity than ICOVIR2, which demonstrates the potentiating effect conferred by the kozak sequence.
FIGURA 7. Efecto de Ia modificación del promotor E2F1 para aumentar su potencia cuando se halla aislado con Ia secuencia DM.FIGURE 7. Effect of the modification of the E2F1 promoter to increase its potency when it is isolated with the DM sequence.
El adenovirus oncolítico ICOVIR7 se distingue de ICOVIR5 por tener el promotor E2F1 modificado. En Ia parte superior de Ia figura se muestra el análisis de Ia expresión de E1a en en Ia línea tumoral 1.36.1.5 de melanoma por western blot. El nivel de expresión de E1a es superior en ICOVIR7 que en ICOVIR5 Io que demuestra el papel potenciador de los sitios de unión a E2F adicionales enThe oncolytic adenovirus ICOVIR7 is distinguished from ICOVIR5 by having the modified E2F1 promoter. In the upper part of the figure the analysis of the expression of E1a in the tumor line 1.36.1.5 of melanoma by western blot is shown. The expression level of E1a is higher in ICOVIR7 than in ICOVIR5 Io which demonstrates the enhancing role of additional E2F binding sites in
ICOVIR7. Abajo se muestra el nivel de producción viral de ICOVIR7, ICOVIR5 e ICOVIR2 en Ia línea tumoral 1.36.1.5. Como control de máxima producción se utiliza el virus AdwtRGD en el que E1a no está regulada. ICOVIR7 es capaz de propagarse con Ia misma potencia que el control AdwtRGD.ICOVIR7. Below is the level of viral production of ICOVIR7, ICOVIR5 and ICOVIR2 in the tumor line 1.36.1.5. The AdwtRGD virus in which E1a is not regulated is used as the maximum production control. ICOVIR7 is capable of propagating with the same power as the AdwtRGD control.
FIGURA 8. Un adenovirus que contiene E1a regulado con el promotor de E2F1 aislado con Ia secuencia DM y Ia secuencia de kozak en el inicio de traducción de E1a puede ser utilizado para el tratamiento de tumores. En Ia parte superior de Ia figura se muestra un experimento in vivo con ratones atímicos de Ia cepa Balb/c que contenían tumores NP9. Un total de 1.2 x 107 células tumorales se inyectaron subcutáneamente en cada flanco posterior del ratón. Después de 15 días los tumores formados (que alcanzaron 70-80 mm3) se distribuyeron en los distintos grupos experimentales (n=10 por grupo). Los tumores se inyectaron con PBS (?) ó 109 partículas virales de ICOVIR-2 (? ) o AdwtRGD (¡ ). La gráfica muestra Ia evolución del volumen tumoral. ICOVIR2 es capaz de inhibir el crecimiento tumoral. La fotografía muestra Ia presencia de virus en el tumor tratado con ICOVIR-2 frente al tratado con PBS. Abajo se muestra el tratamiento por vía sistémica endovenosa con ICOVIR5 de ratones con tumores de melanoma SKMel-28. Tratamiento: PBS («).Una inyección a día 0 de ICOVIR- 5 de 25.1010 partículas virales (vp) (A). Una inyección a día 0 de ICOVIR-5 de 1.1011 vp (4). Una inyección a día 0 de 3.1010 vp y otra de 1.1011 vp separadas entre sí 1 hora (•). Se representa Ia media del crecimiento tumoral de 8-10 tumores/grupo ± S. E. Todos los regímenes de tratamiento con ICOVIR-5 demostraron actividad oncolítica que resulta en una supresión del crecimiento tumoral significativamente distinta al grupo control (PBS), p<0,05. La fotografía muestra Ia presencia de virus en el tumor tratado con ICOVIR5.FIGURE 8. An adenovirus containing E1a regulated with the E2F1 promoter isolated with the DM sequence and the kozak sequence at the start of translation of E1a can be used for the treatment of tumors. In the upper part of the figure, an in vivo experiment is shown with athymic mice of the Balb / c strain containing NP9 tumors. A total of 1.2 x 10 7 tumor cells were injected subcutaneously into each posterior flank of the mouse. After 15 days the tumors formed (which reached 70-80 mm 3 ) were distributed in the different experimental groups (n = 10 per group). Tumors were injected with PBS (?) Or 10 9 viral particles of ICOVIR-2 (?) Or AdwtRGD (¡). The graph shows the evolution of the tumor volume. ICOVIR2 is able to inhibit tumor growth. The photograph shows the presence of virus in the tumor treated with ICOVIR-2 versus the one treated with PBS. The intravenous systemic treatment with ICOVIR5 of mice with SKMel-28 melanoma tumors is shown below. Treatment: PBS («). One injection at day 0 of ICOVIR- 5 of 25.10 10 viral particles (vp) (A). One injection at day 0 of ICOVIR-5 of 1.10 11 vp (4). One injection per day 0 of 3.10 10 vp and another of 1.10 11 vp separated from each other 1 hour (•). The mean tumor growth of 8-10 tumors / group ± SE is represented. All treatment regimens with ICOVIR-5 demonstrated oncolytic activity resulting in a significantly different tumor growth suppression to the control group (PBS), p <0.05 . The photograph shows the presence of virus in the tumor treated with ICOVIR5.
FIGURA 9. Demostración ¡n vivo de Ia reducción de toxicidad tras inyectar endovenosamente adenovirus que contienen E1a regulado con el promotor de E2F1 aislado con la secuencia DM y Ia secuencia de kozak para optimizar Ia traducción de E1a.FIGURE 9. In vivo demonstration of the reduction of toxicity after intravenously injecting adenoviruses containing E1a regulated with the E2F1 promoter isolated with the DM sequence and the kozak sequence to optimize the translation of E1a.
La toxicidad in vivo de un adenovirus que contiene Ia secuencia de kozak en E1a y un promotor E2F1 aislado por DM (ICOVIR5) se comparó con Ia de un virus salvaje y el virus oncolítico que AdD24RGD que expresa E1a bajo el promotor salvaje. Los virus se administraron endovenosamente a distintas dosis (1010, 5x1010 y 1011) en ratones inmunocompetentes Balb/c. A los 3 días postinyección se evaluó parámetros asociados a Ia toxicidad como supervivencia animal (dosis letal 50), peso corporal, nivel de transaminasas séricas, y hematograma. La toxicidad asociada a Ia administración de ICOVIR 5 es muy baja incluso a Ia dosis más elevada. FIGURA 10. Demostración in vivo de reducción de Ia expresión de E1a en tejido no tumoral y de toxicidad tras inyectar endovenosamente adenovirus que contienen E1a regulado con el promotor de E2F1 aislado con Ia secuencia DM y Ia secuencia de kozak para optimizar Ia traducción de E1a.The in vivo toxicity of an adenovirus containing the kozak sequence in E1a and an E2F1 promoter isolated by DM (ICOVIR5) was compared with that of a wild virus and the oncolytic virus that AdD24RGD expressing E1a under the wild promoter. Viruses were administered intravenously at different doses (10 10 , 5x10 10 and 10 11 ) in Balb / c immunocompetent mice. At 3 days post-injection, parameters associated with toxicity such as animal survival (lethal dose 50), body weight, serum transaminase level, and hematogram were evaluated. The toxicity associated with the administration of ICOVIR 5 is very low even at the highest dose. FIGURE 10. In vivo demonstration of reduction of E1a expression in non-tumor tissue and toxicity after intravenously injecting adenoviruses containing E1a regulated with the E2F1 promoter isolated with the DM sequence and the kozak sequence to optimize the translation of E1a.
Los virus indicados se administraron como se describe en Ia figura 9. A los 3 días post-inyección se evaluó Ia expresión de E1a en cortes de hígado por inmunohistoquímica (paneles superiores). E1a no se detecta en los animales inyectados con ICOVIR5. La valoración anatomopatológica de cortes de hígado teñidos con eosina-hematoxilina indica una apariencia normal de los hígados de los ratones inyectados con ICOVIR5 (paneles inferiores).The indicated viruses were administered as described in Figure 9. At 3 days post-injection, the expression of E1a was evaluated in liver sections by immunohistochemistry (upper panels). E1a is not detected in animals injected with ICOVIR5. The pathological evaluation of liver sections stained with eosin-hematoxylin indicates a normal appearance of the livers of mice injected with ICOVIR5 (lower panels).
DESCRlPCiON DETALLADA DE LA INVENCIÓNDETAILED DESCRIPTION OF THE INVENTION
A. Estructura de los adenovirus que contienen E1a regulado con el promotor de E2F1 aislado con Ia secuencia DM, Ia secuencia de kozak para optimizar Ia traducción de E1a y Ia adición de sitios de unión a E2F en el promotor E2F1.A. Structure of adenoviruses containing E1a regulated with the E2F1 promoter isolated with the DM sequence, the kozak sequence to optimize the translation of E1a and the addition of E2F binding sites in the E2F1 promoter.
La presente invención describe el uso en el tratamiento del cáncer de adenovirus que contienen E1a regulado con el promotor de E2F1 aislado con Ia secuencia DM, Ia secuencia de kozak para optimizar Ia traducción de E1a y Ia adición de sitios de unión a E2F en el promotor E2F1. El tratamiento se basa en Ia replicación selectiva de estos virus en tumores que tienen Ia vía del retinoblastoma alterada.The present invention describes the use in the treatment of adenovirus cancer containing E1a regulated with the E2F1 promoter isolated with the DM sequence, the kozak sequence to optimize the translation of E1a and the addition of E2F binding sites in the promoter E2F1. The treatment is based on the selective replication of these viruses in tumors that have the altered retinoblastoma pathway.
La vía del retinoblastoma es el conjunto de interacciones proteicas que se suceden desde Ia membrana celular hasta el núcleo para regular el nivel de fosforilación de Ia proteína del retinoblastoma pRb. El cáncer se caracteriza por una alteración de esta vía de modo que Ia proteína pRb se hiperfosforila o se pierde. Esta alteración de pRb provoca una pérdida de unión de pRb al factor de transcripción E2F y el aumento de E2F libre en el núcleo de las células tumorales. Este factor de transcripción se une a los promotores con sitios de unión específicos de E2F, como el promotor de E2F1 , para aumentar su expresión. El mecanismo de replicación selectiva en tumores de adenovirus que contienen E1a regulado con el promotor de E2F1 aislado con Ia secuencia DM, Ia secuencia de kozak en el inicio de traducción de E1a y Ia adición de sitios de unión a E2F en el promotor E2F1 se basa en que Ia presencia de E2F libre en los tumores activa Ia expresión del promotor E2F1 en este virus y se indica en Ia figura 2 de Ia presente invención. La presencia de Ia secuencia DM permite Ia activación correcta del promotor. La presencia de Ia secuencia de kozak permite Ia síntesis de una cantidad de E1a suficiente para mantener Ia capacidad replicativa y lítica apropiada del virus oncolítico. Asimismo, Ia presencia de sitios de unión a E2F adicionales en el promotor E2F1 permite aumentar el nivel de expresión de E1a para mantener Ia capacidad replicativa y lítica apropiada del virus oncolítico.The retinoblastoma pathway is the set of protein interactions that occur from the cell membrane to the nucleus to regulate the level of phosphorylation of the pRb retinoblastoma protein. The cancer is characterized by an alteration of this pathway so that the pRb protein is hyperphosphorylated or lost. This alteration of pRb causes a loss of binding of pRb to the transcription factor E2F and the increase of free E2F in the nucleus of the tumor cells. This transcription factor binds promoters with specific E2F binding sites, such as the E2F1 promoter, to increase its expression. The mechanism of selective replication in adenovirus tumors containing E1a regulated with the E2F1 promoter isolated with the DM sequence, the kozak sequence at the start of translation of E1a and the addition of E2F binding sites in the E2F1 promoter is based in which the presence of free E2F in tumors activates the expression of the E2F1 promoter in this virus and is indicated in Figure 2 of the present invention. The presence of the DM sequence allows the correct activation of the promoter. The presence of the kozak sequence allows the synthesis of an amount of E1a sufficient to maintain the appropriate replicative and lytic capacity of the oncolytic virus. Likewise, the presence of additional E2F binding sites in the E2F1 promoter allows to increase the level of E1a expression to maintain the appropriate replicative and lytic capacity of the oncolytic virus.
La secuencia humana aislante DM derivada del locus de Ia distrofia miotónica está representada por SEQ. ID 1 (desde Ia posición 368 hasta Ia 1096 de Ia secuencia 1 ). La secuencia DM se caracteriza por contener dos sitios de unión al factor CTCF y un número variable de repeticiones de Ia secuencia CGT que conjuntamente funcionan como un potente aislante de Ia interferencia transcripcional 32. En Ia presente invención Ia secuencia DM actúa para aislar el efecto de los enhancers localizados en Ia secuencia de empaquetamiento del adenovirus próximos al promotor de E1a. El promotor de E1a se substituye por un promotor selectivo de tumores como por ejemplo el promotor E2F1 y, para aislar este promotor de los enhancers presentes en Ia secuencia de empaquetamiento adenoviral, Ia secuencia DM se inserta entre dicha secuencia de empaquetamiento y el promotor E2F1. La secuencia del promotor de E2F1 se muestra en SEQ. ID 1 (desde Ia posición 1283 hasta Ia posición 1564 de Ia secuencia 1). Este promotor se caracteriza por presentar dos sitios de unión a E2F organizados en palíndromas imperfectos y cuatro sitios de unión a Sp1 34. En Ia presente invención Ia secuencia del promotor E2F se modifica por Ia inserción de sitios de unión a E2F adicionales a los que ya existen en el promotor humano salvaje (desde Ia posición 1321 hasta Ia posición 1447 de SEQ. ID 3). Con ello se consigue aumentar tanto Ia represión transcripcional en células normales como Ia activación transcripcional en células tumorales. La traducción de ARNm por los ribosomas eucarióticos se puede optimizar si insertamos Ia secuencia C C A/G C C delante del primer codón ATG traducido 35. Esta secuencia fue identificada por Marylin Kozak y ha recibido el nombre de kozak. En Ia presente invención esta secuencia actúa compensando Ia poca potencia observada experimentalmente cuando un promotor selectivo de tumor como el promotor E2F1 aislado con Ia secuencia DM se utiliza para controlar Ia expresión de E1a (posición 1558 hasta 1562 de SEQ. ID 2).The isolating human DM sequence derived from the locus of myotonic dystrophy is represented by SEQ. ID 1 (from position 368 to 1096 of sequence 1). The DM sequence is characterized by containing two binding sites to the CTCF factor and a variable number of repetitions of the CGT sequence that together function as a potent isolator of transcriptional interference 32 . In the present invention, the DM sequence acts to isolate the effect of enhancers located in the adenovirus packaging sequence close to the E1a promoter. The E1a promoter is replaced by a selective tumor promoter such as the E2F1 promoter and, to isolate this promoter from the enhancers present in the adenoviral packing sequence, the DM sequence is inserted between said packing sequence and the E2F1 promoter. The sequence of the E2F1 promoter is shown in SEQ. ID 1 (from position 1283 to position 1564 of sequence 1). This promoter is characterized by presenting two E2F binding sites organized in imperfect palindromes and four Sp1 binding sites 34 . In the present invention the sequence of the E2F promoter is modified by the insertion of E2F binding sites additional to those already existing in the wild human promoter (from position 1321 to position 1447 of SEQ. ID 3). With this it is possible to increase both the transcriptional repression in normal cells and the transcriptional activation in tumor cells. The translation of mRNA by eukaryotic ribosomes can be optimized if we insert the CCA / GC sequence C in front of the first translated ATG codon 35 . This sequence was identified by Marylin Kozak and has been called kozak. In the present invention this sequence acts by compensating for the little power observed experimentally when a tumor selective promoter such as the E2F1 promoter isolated with the DM sequence is used to control the expression of E1a (position 1558 to 1562 of SEQ. ID 2).
Existen varios métodos para manipular el genoma adenoviral. Los métodos de construcción de adenovirus modificados genéticamente están bien establecidos en el campo de Ia terapia génica y Ia viroterapia con adenovirus 36"41. El método más comúnmente utilizado se basa en construir primero Ia modificación genética deseada en un plásmido que contiene Ia región adenoviral a modificar, para después realizar una recombinación homologa en bacterias con un plásmido que contiene el resto del genoma viral 41. El procedimiento puede ser como sigue:.There are several methods to manipulate the adenoviral genome. The methods of genetically modified adenovirus construction are well established in the field of gene therapy and adenovirus virotherapy 36 "41. The most commonly used method is based on first constructing the desired genetic modification in a plasmid containing the adenoviral region. modify, then perform homologous recombination in bacteria with a plasmid containing the rest of the viral genome 41. The procedure can be as follows:
Otros tipos de mutaciones y manipulaciones genéticas distintas a Ia regulación de Ia expresión de E1a con el promotor de E2F1 aislado con Ia secuencia DM, Ia inserción de Ia secuencia de kozak para optimizar la traducción de E1a y Ia adición de sitios de unión a E2F en el promotor E2F1 descritas en Ia presente invención se han realizado para obtener replicación selectiva en tumoresOther types of mutations and genetic manipulations other than the regulation of the expression of E1a with the E2F1 promoter isolated with the DM sequence, the insertion of the kozak sequence to optimize the translation of E1a and the addition of E2F binding sites in The E2F1 promoter described in the present invention have been performed to obtain selective replication in tumors
1,42-44 frstas pUecjen ser inserciones de otros promotores distintos a E2F1 que son activos en células tumorales y que se usan también para controlar Ia expresión de genes virales. Una realización de Ia presente invención es el uso de Ia secuencia aislante DM y Ia secuencia de kozak en combinación con esos otros promotores. 1.42 - 44 fr s t as p Uec j in being insertions of promoters other than E2F1 that are active in tumor cells and are also used to control the expression of viral genes. An embodiment of the present invention is the use of the DM insulating sequence and the kozak sequence in combination with those other promoters.
Otra modificación descrita para conseguir replicación selectiva en tumores es Ia delección de funciones tempranas de E1 que bloquean Ia vía de RB. La replicación selectiva de dichos mutantes ya ha sido demostrada 9'10. Otros genes virales que interaccionan directamente con pRB como E4 45 y E4orf6/7 46, respectivamente, son candidatos a ser deleccionados para conseguir replicación selectiva en células tumorales.Another modification described to achieve selective replication in tumors is the deletion of early E1 functions that block the RB pathway. The selective replication of these mutants has already been demonstrated 9 '10. Other viral genes that interact directly with pRB such as E4 45 and E4orf6 / 7 46 , respectively, are candidates for deletion to achieve selective replication in tumor cells.
En otra realización de Ia invención los adenovirus con Ia expresión de un gen viral regulada por promotor selectivo aislado con Ia secuencia DM y potenciada con Ia secuencia de kozak pueden contener modificaciones de su cápsida para aumentar su infectividad o dirigirse a receptores presentes en Ia célula tumoral. Las proteínas de Ia cápsida adenoviral se han modificado genéticamente para incluir ligandos que aumentan Ia infectividad o que dirigen el virus a un receptor en Ia célula tumoral 47~53. Dirigir el adenovirus al tumor también se puede conseguir con ligandos bifuncionales que unen al virus por un lado y al receptor tumoral por otro 53"56. Por otro lado, para aumentar Ia persistencia del adenovirus en sangre y con ello aumentar las posibilidades de alcanzar nodulos tumorales diseminados, Ia cápsida puede cubrirse con polímeros como el polietilen-glicol 57'60. Se pueden configurar estas modificaciones en adenovirus que contienen E1a regulado con el promotor de E2F1 aislado con Ia secuencia DM, Ia secuencia de kozak en el inicio de traducción de E1a y Ia adición de sitios de unión a E2F en el promotor E2F1.In another embodiment of the invention, adenoviruses with the expression of a viral gene regulated by selective promoter isolated with the DM sequence and enhanced with the kozak sequence may contain modifications of their capsid to increase their infectivity or target receptors present in Ia tumor cell The adenoviral capsid proteins have been genetically modified to include ligands that increase infectivity or that direct the virus to a receptor in the tumor cell 47 ~ 53 . Directing adenovirus to the tumor can also be achieved with bifunctional ligands that bind the virus on the one hand and the tumor receptor on the other 53 "56. On the other hand, to increase the persistence of adenovirus in blood and thereby increase the chances of reaching nodules Disseminated tumors, the capsid can be covered with polymers such as polyethylene glycol 57.60 . These modifications can be configured in adenoviruses containing E1a regulated with the E2F1 promoter isolated with the DM sequence, the kozak sequence at the start of translation of E1a and the addition of E2F binding sites in the E2F1 promoter.
Otra realización de Ia presente invención son adenovirus que contienenAnother embodiment of the present invention are adenoviruses containing
E1a regulado con el promotor de E2F1 aislado con Ia secuencia DM, Ia secuencia de kozak en el inicio de traducción de E1a y Ia adición de sitios de unión a E2F en el promotor E2F1 pero que derivan de otros serotipos de adenovirus distintos alE1a regulated with the E2F1 promoter isolated with the DM sequence, the kozak sequence at the start of translation of E1a and the addition of E2F binding sites in the E2F1 promoter but derived from other adenovirus serotypes other than
Ad5.Ad5
Otra realización de Ia presente invención se refiere a adenovirus que contienen E1a regulado con el promotor de E2F1 aislado con Ia secuencia DM, Ia secuencia de kozak en el inicio de traducción de E1a y Ia adición de sitios de unión a E2F en el promotor E2F1 y que a su vez contienen otros genes para aumentar su citotoxicidad sobre células tumorales como el gen de Ia timidina quinasa, citosina deaminasa, genes proapoptóticos, inmunoestimuladores o supresores tumorales.Another embodiment of the present invention relates to adenoviruses containing E1a regulated with the E2F1 promoter isolated with the DM sequence, the kozak sequence at the start of translation of E1a and the addition of E2F binding sites in the E2F1 promoter and which in turn contain other genes to increase their cytotoxicity on tumor cells such as the thymidine kinase gene, cytosine deaminase, proapoptotic genes, immunostimulators or tumor suppressors.
B. Producción, purificación y formulación de adenovirus que contienen E1a regulado con el promotor de E2F1 aislado con Ia secuencia DM, Ia secuencia de kozak en el inicio de traducción de E1a y Ia adición de sitios de unión a E2F en el promotor E2F1.B. Production, purification and formulation of adenovirus containing E1a regulated with the E2F1 promoter isolated with the DM sequence, the kozak sequence at the start of translation of E1a and the addition of E2F binding sites in the E2F1 promoter.
Los adenovirus descritos en Ia presente invención se propagan siguiendo métodos estándar en los campos de Ia adenovirología y los vectores adenovirales 36'37. El método preferido de propagación es por infección de una línea celular permisiva a Ia replicación de adenovirus que contienen E1a regulado con el promotor de E2F1 aislado con Ia secuencia DM, Ia secuencia de kozak en el inicio de traducción de E1a y Ia adición de sitios de unión a E2F en el promotor E2F1. La línea de adenocarcinoma pulmonar A549 es un ejemplo de dicha línea. La propagación se realiza por ejemplo del siguiente modo: Las células A549 se crecen sobre placas de cultivo celular de plástico y se infectan usando 50 partículas virales por célula. Dos días después el efecto citopático que refleja Ia producción de virus se observa como un arracimamiento de las células. Las células se recogen y se almacenan en tubos. Después de una centrifugación a 1000g durante 5 minutos, el precipitado celular se congela y descongela tres veces para romper las células. El extracto celular resultante se centrifuga a 100Og durante 5 minutos y el sobrenadante con virus se carga encima de un gradiente de cloruro de cesio y se centrifuga durante 1 hora a 35.00Og. La banda de virus en el gradiente se carga de nuevo sobre otro gradiente de cloruro de cesio y se centrifuga durante 16 horas a 35.00Og. La banda de virus se recoge y se dializa frente a PBS-10% glicerol. El virus dializado se alícuota y almacena a -8O0C. La cuantificación de número de partículas y unidades formadoras de placa se realiza siguiendo protocolos estándar 39. Tampón fosfato salino con glicerol al 10% es una formulación estándar para el almacenamiento de adenovirus. Sin embargo se han descrito nuevas formulaciones que mejoran Ia estabilidad del virus 61>62.The adenoviruses described in the present invention are propagated following standard methods in the fields of adenovirology and adenoviral vectors 36 '37 . The preferred method of propagation is by infection of a permissive cell line to the replication of adenovirus containing E1a regulated with the E2F1 promoter isolated with the DM sequence, the kozak sequence at the start of translation of E1a and the addition of sites of E2F binding in the E2F1 promoter. The A549 pulmonary adenocarcinoma line is an example of that line. Propagation is carried out, for example, as follows: A549 cells are grown on plastic cell culture plates and infected using 50 viral particles per cell. Two days later the cytopathic effect that reflects the production of virus is observed as a clustering of the cells. The cells are collected and stored in tubes. After centrifugation at 1000g for 5 minutes, the cell precipitate freezes and thaws three times to break the cells. The resulting cell extract is centrifuged at 100Og for 5 minutes and the virus supernatant is loaded on top of a cesium chloride gradient and centrifuged for 1 hour at 35.00Og. The virus band in the gradient is reloaded onto another gradient of cesium chloride and centrifuged for 16 hours at 35.00Og. The virus band is collected and dialyzed against PBS-10% glycerol. The dialyzed virus is aliquot and stored at -8O 0 C. The quantification of the number of particles and plaque forming units is performed following standard protocols 39 . Phosphate buffered saline with 10% glycerol is a standard formulation for adenovirus storage. However, new formulations have been described that improve the stability of virus 61> 62 .
C. Utilización de adenovirus que contienen E1a regulado con el promotor de E2F1 aislado con Ia secuencia DM, Ia secuencia de kozak en el inicio de traducción de E1a y Ia adición de sitios de unión a E2F en el promotor E2F1 para el tratamiento del cáncer.C. Use of adenoviruses containing E1a regulated with the E2F1 promoter isolated with the DM sequence, the kozak sequence at the start of translation of E1a and the addition of E2F binding sites in the E2F1 promoter for the treatment of cancer.
La presente invención describe el uso de adenovirus que contienen E1a regulado con el promotor de E2F1 aislado con Ia secuencia DM, Ia secuencia de kozak en el inicio de traducción de E1a y Ia adición de sitios de unión a E2F en el promotor E2F1 para tratar el cáncer. El tratamiento se basa en Ia replicaclón selectiva de estos virus en células con una vía de RB activa.The present invention describes the use of adenoviruses containing E1a regulated with the E2F1 promoter isolated with the DM sequence, the kozak sequence at the start of translation of E1a and the addition of E2F binding sites in the E2F1 promoter to treat cancer. The treatment is based on the selective replication of these viruses in cells with an active RB pathway.
Los protocolos para usar los virus descritos en Ia presente invención en el tratamiento del cáncer siguen los mismos procedimientos que los usados en los campos de viroterapia con adenovirus y terapia génica con adenovirus. Existe una amplia experiencia en el uso de adenovirus no replicativos y replicativos en el campo de Ia terapia génica. En particular adenovirus con mecanismos de replicación selectiva distintos al propuesto en Ia presente invención han sido usados para tratar el cáncer 9-37-63-68. Existen numerosas publicaciones de tratamiento de células tumorales en cultivo, en modelos animales y en ensayos clínicos con pacientes. Para el tratamiento de células en cultivos in vitro el adenovirus purificado en cualquiera de las formulaciones descritas más arriba se añade al medio de cultivo para Ia infección de las células tumorales. Para tratar tumores en modelos animales o en pacientes humanos el adenovirus se puede administrar loco-regionalmente por inyección en el tumor o en una cavidad corporal donde se localiza el tumor, o bien sistémicamente por inyección en el torrente sanguíneo. Como se ha practicado con otros adenovirus de replicación selectiva, el tratamiento de tumores con los adenovirus descritos objeto de Ia presente invención se puede combinar con otras modalidades terapéuticas como Ia quimioterapia o radioterapia.The protocols for using the viruses described in the present invention in the treatment of cancer follow the same procedures as those used in the fields of adenovirus virotherapy and adenovirus gene therapy. There is extensive experience in the use of non-replicative and replicative adenoviruses in the field of gene therapy. In particular , adenoviruses with selective replication of various proposed in the present invention have been used to treat cancer 9-37 - 63-68. There are numerous publications on the treatment of tumor cells in culture, in animal models and in clinical trials with patients. For the treatment of cells in in vitro cultures, the purified adenovirus in any of the formulations described above is added to the culture medium for the infection of the tumor cells. To treat tumors in animal models or in human patients, the adenovirus can be administered loco-regionally by injection into the tumor or into a body cavity where the tumor is located, or systemically by injection into the bloodstream. As it has been practiced with other adenoviruses of selective replication, the treatment of tumors with the adenoviruses described object of the present invention can be combined with other therapeutic modalities such as chemotherapy or radiotherapy.
EJEMPLOSEXAMPLES
EJEMPLO 1EXAMPLE 1
Un adenovirus oncolítico con E1a regulada con el promotor de E2F1 aislado con Ia secuencia DM expresa E1a v se replica selectivamente en células tumorales.An oncolytic adenovirus with E1a regulated with the E2F1 promoter isolated with the DM sequence expresses E1a v selectively replicates in tumor cells.
Se construyó un adenovirus con E1a regulada con el promotor de E2F1 aislado con Ia secuencia DM del siguiente modo: Para generar ICOVIR-1 (Ad- E2F-Δ24RGD), el promotor E2F1 humano se obtuvo por PCR de células mononucleares de sangre periférica humana usando oligonucleótidos que amplifican desde el par de bases -218 al +51 del promotor E2F-1 (Ia posición +1 indica el inicio de transcripción). Los oligonucleótidos contenían dianas de restricción Kpn\ y HindW para Ia clonación en el piásmido pGL3 (Promega, South Hampton, UK). El piásmido resultante se denominó pGL3-E2F. A partir de este se obtuvo pE2F- Δ24 por recombinación con un piásmido que contiene las 5,766 pares de bases del extremo izquierdo del genoma adenoviral exceptuando los nucleótidos (nt) 122 a 129 de E1a (derivado de pXC1-Δ24 con un sitio Hind\\\ entre nt 348 y el nt 522 del genoma de Ad5 9). pE2F- Δ24 se recombinó con pShuttle 41 para obtener pShuttle-E2F- Δ24. Este piásmido se linearizó con Pme\ y se recombinó con pVK503 (que contiene Ia secuencia de Ad5 con Ia fibra modificada con RGD 69) para generar el piásmido pAd-E2F-Δ24RGD ó pICOVIR- 1. La combinación del promotor E2F1 y otras modificaciones descritas en esta invención con Ia mutación de E1a denominada Δ24 y Ia inserción del péptido RGD en Ia fibra se realizó para demostrar que las modificaciones presentadas en esta invención aumentan Ia selectividad y potencia oncolítica de un virus reconocido como selectivo de Ia vía de Rb y potente en el campo de Ia oncolisis (adenovirus Ad-Δ24RGD 70). El virus ICOVIR1 se generó por digestión con Pací de este piásmido y transfección en células HEK293. Un protocolo paralelo se utilizó para generar ICOVIR-2 (Ad-DM-E2F-Δ24RGD). La secuencia aislante DM-1 se obtuvo de PCR de células mononucleares de sangre periférica humana usando oligonucleotidos que amplifican desde el nt 13006 al nt 13474 del locus DM1 (secuencia publicada en GenBank con número L08835). Los oligonucleotidos de Ia PCR se diseñaron para incorporar sitios Xho I flanqueantes. El DM-1 se subclonó en Xhol de pShuttle-E2F-Δ24 arriba descrito para obtener pShuttle-DM- E2F-Δ24. La correcta orientación del fragmento DM1 se verificó por restricción con βaA7?H1 , HinúlW, Xhol y Sma\. pShuttle-DM-E2F-Δ24 se recombinó con pVK503 para generar plCOVIR2. El virus ICOVIR2 se generó por digestión con Pací de este piásmido y transfección en células HEK293. ICOVIR1 e ICOVIR2 se propagaron en Ia línea A549 y se purificaron por métodos descritos en terapia génica y viroterapia 36. La estructura correcta de los genomas de ICOVIR-1 e ICOVIR-2 se verificó por restricción con Kpn\ y H/ndlII respectivamente. Adicionalmente se secuenció Ia región DM-1 , el promotor E2F, Ia mutación E1A- Δ24 y Ia región de Ia fibra que contiene RGD. Los oligonucleotidos usados para estas secuenciaciones son: DMI-Up (δ'-GGGCAGATGGAGGGCCTTTTATTC-S1), E2F-Up (5'-GTGTTACTCATAGCGCGTAA-3'), Δ24-down (5'-An adenovirus with E1a regulated with the E2F1 promoter isolated with the DM sequence was constructed as follows: To generate ICOVIR-1 (Ad-E2F-Δ24RGD), the human E2F1 promoter was obtained by PCR of human peripheral blood mononuclear cells using oligonucleotides that amplify from base pair -218 to +51 of the E2F-1 promoter (position +1 indicates the start of transcription). The oligonucleotides contained targets of Kpn \ and HindW restriction for cloning in the plasmid pGL3 (Promega, South Hampton, UK). The resulting plasmid was named pGL3-E2F. From this, pE2F-Δ24 was obtained by recombination with a pyramid containing 5,766 base pairs from the left end of the adenoviral genome except nucleotides (nt) 122 to 129 of E1a (derived from pXC1-Δ24 with a Hind site \\ between nt 348 and nt 522 of the genome of Ad5 9 ). pE2F- Δ24 was recombined with pShuttle 41 to obtain pShuttle-E2F- Δ24. This plasmid was linearized with Pme \ and recombined with pVK503 (which contains the Ad5 sequence with the fiber modified with RGD 69 ) to generate the plasmid pAd-E2F-Δ24RGD or pICOVIR-1. The combination of the E2F1 promoter and other modifications described In this invention, with the mutation of E1a called Δ24 and the insertion of the RGD peptide into the fiber, it was performed to demonstrate that the modifications presented in this invention increase the selectivity and oncolytic potency of a virus recognized as selective of the Rb pathway and potent in the field of oncolysis (adenovirus Ad-Δ24RGD 70 ). The ICOVIR1 virus was generated by digestion with Pací of this pyramid and transfection in HEK293 cells. A parallel protocol was used to generate ICOVIR-2 (Ad-DM-E2F-Δ24RGD). The DM-1 isolating sequence was obtained from PCR of human peripheral blood mononuclear cells using oligonucleotides that amplify from nt 13006 to nt 13474 of the DM1 locus (sequence published in GenBank with number L08835). The oligonucleotides of the PCR were designed to incorporate flanking Xho I sites. The DM-1 was subcloned into Xhol of pShuttle-E2F-Δ24 described above to obtain pShuttle-DM-E2F-Δ24. The correct orientation of the DM1 fragment was verified by restriction with βaA7? H1, HinulW, Xhol and Sma \. pShuttle-DM-E2F-Δ24 was recombined with pVK503 to generate plCOVIR2. The ICOVIR2 virus was generated by Paci digestion of this pyramid and transfection in HEK293 cells. ICOVIR1 and ICOVIR2 were propagated in the A549 line and purified by methods described in gene therapy and virotherapy 36 . The correct structure of the genomes of ICOVIR-1 and ICOVIR-2 was verified by restriction with Kpn \ and H / ndlII respectively. Additionally, the DM-1 region, the E2F promoter, the E1A-Δ24 mutation and the fiber region containing RGD were sequenced. The oligonucleotides used for these sequencing are: DMI-Up (δ'-GGGCAGATGGAGGGCCTTTTATTC-S 1 ), E2F-Up (5'-GTGTTACTCATAGCGCGTAA-3 '), Δ24-down (5'-
CCTCCGGTGATAATGACAAG-3') y FiberUp (δ'-CAAACGCTGTTGGATTTATG- 3'). Las secuencias obtenidas se muestran en SEQ. ID 1.CCTCCGGTGATAATGACAAG-3 ') and FiberUp (δ'-CAAACGCTGTTGGATTTATG-3'). The sequences obtained are shown in SEQ. ID 1.
Para demostrar que un adenovirus oncolítico con E1a regulada con el promotor de E2F1 aislado con Ia secuencia DM expresa E1a selectivamente en células tumorales se procedió a infectar cultivos celulares de células normales (hepatocitos murinos y humanos, fibroblastos humanos y células endoteliales humanas HUVEC) y tumorales (células de carcinoma de páncreas NP9, carcinoma de pulmón A549, carcinomas de cabeza y cuello FaDu y SCC25, y melanoma SK-Mel-28 y 1.36.1.5) con ICOVIR1 e ICOVIR2 usando multiplicidad de infección que permitía más del 80% de infección. Después de 20 horas postinfección se usaron las células en tampón de lisis (400 mM NaCI, 1 mM EDTA, 5 mM NaF, 10% glicerol, 1mM orthovanadato de sodio, 0,5% Nonidet P-40, 100 ug/ml fluoruro de phenylmethylsulfonyl , 1 ug/ml leupeptina y 10 ug/ml aprotinina en 1OmM Tris-HCI (pH 7.4)) durante 1 h a 40C. El lisado se centrifugó a 1400Og, y el sobrenadante con proteínas se separó por electroforesis en 10% SDS-PAGE (25 ug/carril, determinados por Bradford, BioRad, CA, EEUU) y se transfirió a nitro-celulosa (Schleicher and Schuell, Dassel, Alemania). La membrana se bloqueó con 5% de leche desnatada, 0.05% Tween 20, 0.9% NaCI en 5OmM Tris (pH 7.5), y se incubó 16h a 40C con un anticuerpo policlonal anti-adenovirus-2 E1a (clon 13 S-5, Santa Cruz Biotechnology Inc., Santa Cruz, CA, EEUU). E1a se reveló con un anticuerpo secundario anti-conejo IgG (DAKO A/S) conjugado a peroxidasa y el protocolo de quimioluminiscencia de "Amersham's Enhanced Chemioluminiscence" (Amersham, Arlington Heights, IL, EUU). El resultado se muestra en Ia figura 3 de Ia presente invención. Se demuestra que Ia presencia del promotor E2F1 (ICOVIR1) es capaz de disminuir Ia expresión de E1a en células normales. Pero Ia secuencia DM confiere un mayor control de Ia expresión de E1a por el promotor E2F (ICOVIR2). En células tumorales tanto ICOVIR1 como ICOVIR2 son capaces de expresar E1a, pero es importante señalar que en algunas líneas tumorales como FaDu, SCC25 y SKMel-28 Ia expresión de E1a es menor que Ia obtenida con el adenovirus salvaje y el oncolítico AdD24RGD en donde E1a no está regulada por E2F1. Ello indica que el promotor de E2F1 aislado o no con DM no tiene Ia potencia necesaria para permitir un nivel de expresión de E1 a en células tumorales comparable al adenovirus salvaje.To demonstrate that an oncolytic adenovirus with E1a regulated with the E2F1 promoter isolated with the DM sequence expressly expresses E1a in tumor cells, normal cell cultures (murine and human hepatocytes, human fibroblasts and human HUVEC endothelial cells) and tumor cells were infected (NP9 pancreatic carcinoma cells, A549 lung carcinoma, FaDu and SCC25 head and neck carcinomas, and SK-Mel-28 and 1.36.1.5 melanoma) with ICOVIR1 and ICOVIR2 using multiplicity of infection that allowed more than 80% infection . After 20 hours post-infection, the cells were used in lysis buffer (400 mM NaCI, 1 mM EDTA, 5 mM NaF, 10% glycerol, 1mM sodium orthovanadate, 0.5% Nonidet P-40, 100 ug / ml fluoride phenylmethylsulfonyl, 1 ug / ml leupeptin and 10 ug / ml aprotinin in 1OmM Tris-HCI (pH 7.4)) for 1 h at 4 0 C. The lysate was centrifuged at 1400Og, and the protein supernatant was electrophoresed in 10% SDS -PAGE (25 ug / lane, determined by Bradford, BioRad, CA, USA) and transferred to nitrocellulose (Schleicher and Schuell, Dassel, Germany). The membrane was blocked with 5% skim milk, 0.05% Tween 20, 0.9% NaCI in 5OmM Tris (pH 7.5), and incubated 16h at 4 0 C with a polyclonal anti-adenovirus-2 E1a antibody (clone 13 S- 5, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). E1a was revealed with a secondary anti-rabbit IgG antibody (DAKO A / S) conjugated to peroxidase and the chemiluminescence protocol of "Amersham's Enhanced Chemioluminescence" (Amersham, Arlington Heights, IL, EUU). The result is shown in Figure 3 of the present invention. It is demonstrated that the presence of the E2F1 promoter (ICOVIR1) is capable of decreasing the expression of E1a in normal cells. However, the DM sequence confers greater control of the expression of E1a by the E2F promoter (ICOVIR2). In tumor cells both ICOVIR1 and ICOVIR2 are capable of expressing E1a, but it is important to note that in some tumor lines such as FaDu, SCC25 and SKMel-28 the expression of E1a is less than that obtained with the wild adenovirus and the oncolytic AdD24RGD where E1a It is not regulated by E2F1. This indicates that the E2F1 promoter isolated or not with DM does not have the power necessary to allow a level of expression of E1 a in tumor cells comparable to wild adenovirus.
Para demostrar que un adenovirus oncolítico con E1a regulada con el promotor de E2F1 aislado con Ia secuencia DM se replica selectivamente en células tumorales se procedió a infectar las células con ICOVIR1 e ICOVIR2 como se describe en el párrafo anterior. Cinco días post-infección se recolectaron las células y sus medios de cultivo y se sometieron a tres ciclos de congelación- descongelación para liberar el virus. La cantidad de virus en el extracto celular se determinó por infección en HEK293 y tinción anti-hexón utilizando el anticuerpo monoclonal 2Hx-2 (ATCC) y un anticuerpo secundario Alexa 488 anti-lgG de ratón (Molecular Probes, Eugene, OR). El resultado se muestra en Ia figura 4. La presencia del promotor E2F1 en ICOVIR1 reduce Ia replicación viral en células normales (fibroblastos y HUVEC). Sin embargo, Ia secuencia aislante en ICOVIR2 resulta en una menor replicación viral. En ciertas líneas células tumorales como A549, ICOVIR1 e ICOVIR2 muestran un nivel de replicación similar al adenovirus salvaje Adwt pero en Ia mayoría de líneas tumorales su capacidad replicativa es menor que Ia de Adwt.To demonstrate that an oncolytic adenovirus with E1a regulated with the E2F1 promoter isolated with the DM sequence is selectively replicated in tumor cells, the cells were infected with ICOVIR1 and ICOVIR2 as described in the previous paragraph. Five days post-infection the cells and their culture media were collected and subjected to three freeze-thaw cycles to release the virus. The amount of virus in the cell extract was determined by infection in HEK293 and anti-hexon staining using the 2Hx-2 monoclonal antibody (ATCC) and a secondary Alexa 488 anti-mouse IgG antibody (Molecular Probes, Eugene, OR). The result is shown in Figure 4. The presence of the E2F1 promoter in ICOVIR1 reduces viral replication in normal cells (fibroblasts and HUVEC). However, the isolating sequence in ICOVIR2 results in a lower viral replication. In certain lines, tumor cells such as A549, ICOVIR1 and ICOVIR2 show a level of replication similar to the Adwt wild adenovirus but in the majority of tumor lines its replicative capacity is lower than that of Adwt.
EJEMPLO 2 La secuencia de kozak permite aumentar Ia expresión de E1a un adenovirus oncolítico en el que Ia expresión de E1a está regulada con el promotor de E2F1 aislado con Ia secuencia DM.EXAMPLE 2 The kozak sequence allows to increase the expression of E1a an oncolytic adenovirus in which the expression of E1a is regulated with the E2F1 promoter isolated with the DM sequence.
Se construyó un adenovirus oncolítico con E1a regulada con el promotor de E2F1 aislado con Ia secuencia DM y con Ia secuencia de kozak para aumentar su traducción. Para ello un fragmento de DNA que contenía Ia secuencia DM, el promotor E2F1 y E1a fue aislado del pShuttle-DM-E2F-D24 descrito en el ejemplo 1 por restricción con Kpn1 y subclonado en pGEM3Z (Promega) obteniéndose el plásmido pGEM-E2F-d24. Este plásmido se utilizó para reemplazar el inicio de traducción de E1a utilizando oligonucleótidos con Ia secuencia de kozak obteniéndose pGEM-E2F-KD24. El fragmento Kpn1 así modificado se reclonó en Kpn1 de pShuttle-DM-E2F-D24 para obtener pShuttle- DM-E2F-KD24. Finalmente pShuttle-DM-E2F-KD24 se recombinó con pVK503 para obtener pICOVIRδ. El virus ICOVIR5 se generó por digestión con Pac\ de este plásmido y transfección en células HEK293. ICOVIR5 se propagó en Ia línea A549 y se purificó por métodos descritos en terapia génica y viroterapia 36. Su estructura se presenta en Ia figura 1 de Ia presente invención. La secuencia correcta del promotor y E1a se verificó por restricción y secuenciación. La secuencia obtenida se muestra en SEQ. ID 2.An oncolytic adenovirus was constructed with E1a regulated with the E2F1 promoter isolated with the DM sequence and with the kozak sequence to increase its translation. For this, a DNA fragment containing the DM sequence, the E2F1 and E1a promoter was isolated from the pShuttle-DM-E2F-D24 described in example 1 by restriction with Kpn1 and subcloned into pGEM3Z (Promega) obtaining plasmid pGEM-E2F- d24. This plasmid was used to replace the translation start of E1a using oligonucleotides with the kozak sequence obtaining pGEM-E2F-KD24. The Kpn1 fragment thus modified was reclassified in Kpn1 of pShuttle-DM-E2F-D24 to obtain pShuttle- DM-E2F-KD24. Finally pShuttle-DM-E2F-KD24 was recombined with pVK503 to obtain pICOVIRδ. The ICOVIR5 virus was generated by digestion with Pac \ of this plasmid and transfection in HEK293 cells. ICOVIR5 was propagated in the A549 line and purified by methods described in gene therapy and virotherapy 36 . Its structure is presented in Figure 1 of the present invention. The correct sequence of the promoter and E1a was verified by restriction and sequencing. The sequence obtained is shown in SEQ. ID 2.
Para demostrar que E1a se expresa condicionalmente en células tumorales cuando su expresión está regulada con el promotor de E2F1 aislado con Ia secuencia DM y además su traducción se optimiza con Ia secuencia de kozak, se procedió a analizar Ia expresión de E1a como se describe en el ejemplo 1. En este caso se incluyó en adenovirus oncolítico ICOVIR5, que se distingue de ICOVIR2 por contener Ia secuencia de kozak en el inicio de traducción de E1a. Los resultados se muestran en Ia figura 5 de Ia presente invención. En células normales ICOVIR5 no expresa E1a por presentar el promotor E2F aislado con DM. En células tumorales el nivel de expresión de E1a es superior en ICOVIR5 que en ICOVIR2, Io que demuestra el efecto de Ia secuencia de kozak para aumentar Ia potencia del promotor aislado con DM.To demonstrate that E1a is conditionally expressed in tumor cells when its expression is regulated with the E2F1 promoter isolated with the DM sequence and in addition its translation is optimized with the kozak sequence, the expression of E1a was analyzed as described in the Example 1. In this case, ICOVIR5 oncolytic adenovirus was included, which is distinguished from ICOVIR2 because it contains the kozak sequence at the beginning of E1a translation. The results are shown in Figure 5 of the present invention. In normal cells ICOVIR5 does not express E1a by presenting the E2F promoter isolated with DM. In tumor cells, the expression level of E1a is higher in ICOVIR5 than in ICOVIR2, which demonstrates the effect of the kozak sequence to increase the potency of the promoter isolated with DM.
EJEMPLO 3EXAMPLE 3
La secuencia de kozak permite aumentar Ia potencia oncolítica de un adenovirus en el que Ia expresión de E1a está regulada con el promotor de E2F1 aislado con Ia secuencia DM.The kozak sequence allows to increase the oncolytic potency of an adenovirus in which the expression of E1a is regulated with the E2F1 promoter isolated with the DM sequence.
Se cultivaron en pocilios de placas de 96 pocilios las células de las líneas tumorales SKMel-28 y FaDu en las cuales se había visto una disminución de Ia capacidad replicativa de ICOVIR2 (como se ha descrito en el ejemplo 1 y figura 4). Estas células se infectaron con cantidades crecientes de ICOVIR5, ICOVIR2 y AdwtRGD (este último utilizado como control de máxima potencia lítica). Cinco días post-infección se evaluó espectrofotométricamente Ia cantidad de proteína como reflejo de Ia supervivencia celular. Los resultados se muestran en Ia figura 6 de Ia presente invención. La capacidad lítica de ICOVIR5 en SKMel-28 es Ia misma que Ia de AdwtRGD y superior a ICOVIR2. En FaDu es también superior a ICOVIR2 aunque no alcanza el nivel del AdwtRGD.The cells of the SKMel-28 and FaDu tumor lines were cultured in 96-well plates in which a decrease in the replicative capacity of ICOVIR2 had been seen (as described in Example 1 and Figure 4). These cells were infected with increasing amounts of ICOVIR5, ICOVIR2 and AdwtRGD (the latter used as a control of maximum lithic power). Five days post-infection the amount of protein was spectrophotometrically evaluated as a reflection of cell survival. The results are shown in Figure 6 of the present invention. The lithic capacity of ICOVIR5 in SKMel-28 is the same as that of AdwtRGD and greater than ICOVIR2. In FaDu it is also superior to ICOVIR2 although it does not reach the level of AdwtRGD.
EJEMPLO 4EXAMPLE 4
La modificación del promotor E2F1 por inserción de sitios de unión a E2F permite aumentar en células tumorales Ia expresión de E1a cuando E1a está regulada con el promotor de E2F1 aislado con Ia secuencia DM y además su traducción se optimiza con Ia secuencia de kozak.The modification of the E2F1 promoter by insertion of E2F binding sites allows the expression of E1a to be increased in tumor cells when E1a is regulated with the E2F1 promoter isolated with the DM sequence and also its translation is optimized with the kozak sequence.
Se construyó un adenovirus oncolítico con E1a regulada con un promotor de E2F1 modificado por Ia inserción de cuatro sitios de unión a E2F. Para ello en el plásmido pGEM-E2FKE1ad24 descrito en el ejemplo 2 se introdujo mediante mutagénesis dirigida una diana para BsiWI en el promotor de E2F1 (posición 1326). En este sitio BsiWI se ligaron dos copias de oligonucleótidos con Ia secuencia palindrómica de unión a E2F y que tenían extremos compatibles con BsiWI. El promotor así modificado se sublconó en Kpn1 de pShuttle-DM-E2F-D24 para obtener pShDME2FBsiE2F2KE1ad24. Por recombinación homologa de este plásmido con un genoma AdwtRGD se obtuvo el plásmido plCOVIR7. El virus ICOVIR7 se generó por digestión con Pací de este plásmido y transfección en células HEK293. ICOVIR7 se propagó en Ia línea A549 y se purificó por métodos descritos en terapia génica y viroterapia 36. Su estructura se presenta en Ia figura 1 de Ia presente invención. La secuencia correcta del promotor y E1a se verificó por restricción y secuenciación. La secuencia obtenida se muestra en SEQ. ID 3. Para demostrar el papel del promotor E2F1 modificado en el contexto de aislamiento obtenido con DM se procedió a analizar Ia expresión de E1a en Ia línea tumoral 1.36.1.5 de melanoma por western blot como se describe en el ejemplo 1. El adenovirus oncolítico ICOVIR7 se distingue de ICOVIR5 por tener el promotor E2F1 modificado. Los resultados se muestran en Ia figura 7 de Ia presente invención. El nivel de expresión de E1a es superior en ICOVIR7 Io que demuestra el papel potenciador de los sitios de unión a E2F adicionales en ICOVIR7. Además Ia expresión de E1a es mayor en ICOVIR5 que en ICOVIR2, Io que demuestra de nuevo el efecto de Ia secuencia de kozak para aumentar Ia potencia del promotor aislado con DM.An oncolytic adenovirus with E1a regulated with an E2F1 promoter modified by the insertion of four E2F binding sites was constructed. For this, plasmid pGEM-E2FKE1ad24 described in example 2 was introduced by means of directed mutagenesis for BsiWI in the E2F1 promoter (position 1326). In this BsiWI site, two copies of oligonucleotides were ligated with the palindromic sequence of E2F binding and which had ends compatible with BsiWI. The promoter thus modified was sublconized in Kpn1 of pShuttle-DM-E2F-D24 to obtain pShDME2FBsiE2F2KE1ad24. By homologous recombination of this plasmid with an AdwtRGD genome, plasmid plCOVIR7 was obtained. The ICOVIR7 virus was generated by Paci digestion of this plasmid and transfection into HEK293 cells. ICOVIR7 was propagated in line A549 and purified by methods described in gene therapy and virotherapy 36 . Its structure is presented in Figure 1 of the present invention. The correct sequence of the promoter and E1a was verified by restriction and sequencing. The sequence obtained is shown in SEQ. ID 3. To demonstrate the role of the modified E2F1 promoter in the context of isolation obtained with DM, the expression of E1a in the 1.36.1.5 tumor line of melanoma by western blot was analyzed as described in example 1. Oncolytic adenovirus ICOVIR7 is distinguished from ICOVIR5 by having the modified E2F1 promoter. The results are shown in Figure 7 of the present invention. The expression level of E1a is higher in ICOVIR7 which demonstrates the potentiating role of additional E2F binding sites in ICOVIR7. In addition, the expression of E1a is higher in ICOVIR5 than in ICOVIR2, Io which demonstrates again the effect of the kozak sequence to increase the potency of the promoter isolated with DM.
EJEMPLO 5 Un adenovirus que contiene E1a regulado con el promotor de E2F1 aislado con Ia secuencia DM v Ia secuencia de kozak en el inicio de traducción de E1a puede ser utilizado para tratar eficazmente tumores.EXAMPLE 5 An adenovirus containing E1a regulated with the E2F1 promoter isolated with the DM sequence and the kozak sequence at the start of translation of E1a can be used to effectively treat tumors.
Se realizó un experimento in vivo con ratones atímicos de Ia cepa Balb/c que contenían tumores NP9. Un total de 1.2 x 107 células tumorales de Ia línea SKMel-28 se inyectaron subcutáneamente en cada flanco posterior del ratón. Después de 15 días los ratones con tumores formados (que alcanzan 70-80 mm3) se distribuyeron en los distintos grupos experimentales (n=10 por grupo). Los tumores del grupo control recibieron dos inyecciones intratumorales de tampón salino (2 x 10 μl). Los del grupo tratado con icovirδ recibieron dos inyecciones intratumorales (2 x 10 μl) de icovirδ (109 partículas virales por tumor). Se midieron los tumores cada dos días y su volumen se estimó según Ia fórmula: V (mm3)= A (mm) x B2 (mm2) x π/6, en donde B es Ia longitud transversal. La figura 8 muestra el volumen tumoral respecto al inicio del tratamiento (día 0). Los resultados se presentan como media + S.E.M. La existencia de diferencias significativas entre los resultados se calculó usando un ensayo no paramétrico de datos no apareados de Mann-Whitney. Las curvas de crecimiento se compararon usando un análisis de Ia variancia. Los resultados se consideraron significativos si p < 0.05. Los cálculos se realizaron con el paquete estadístico SPSS (SPSS Inc., Chicago, IL). Existe una diferencia significativa entre el tamaño tumoral a días 16 y 21.An in vivo experiment was performed with athymic mice of the Balb / c strain that contained NP9 tumors. A total of 1.2 x 10 7 tumor cells of the SKMel-28 line were injected subcutaneously in each posterior flank of the mouse. After 15 days the mice with tumors formed (reaching 70-80 mm 3 ) were distributed in the different experimental groups (n = 10 per group). Tumors in the control group received two intratumoral injections of saline buffer (2 x 10 μl). Those in the icovirδ-treated group received two intratumoral injections (2 x 10 μl) of icovirδ (10 9 viral particles per tumor). Tumors were measured every two days and their volume was estimated according to the formula: V (mm 3 ) = A (mm) x B 2 (mm 2 ) x π / 6, where B is the transverse length. Figure 8 shows the tumor volume with respect to the start of treatment (day 0). The results are presented as mean + SEM The existence of significant differences between the results was calculated using a non-parametric trial of unpaired Mann-Whitney data. The growth curves were compared using an analysis of the variance. The results were considered significant if p <0.05. Calculations were performed with the SPSS statistical package (SPSS Inc., Chicago, IL). There is a significant difference between tumor size on days 16 and 21.
En otro experimento el tratamiento se realizó por inyección sistémica de ICOVIR5. Tumores de Ia línea celular de melanoma humana SKMel-28 (1.107 células/tumor) se implantaron en ratones atímicos BaIb C nu/nu, y una vez establecidos fueron tratados por administración en Ia vena de Ia cola con PBS, con una única inyección a día 0 de ICOVIR-5 de 2,5.1010 partículas virales (vp) ó de 1.1011 vp , o con una inyección de 3.1010 vp y otra de 1.1011 vp separadas entre sí 1 hora. Los resultados se muestran en Ia parte inferior de Ia figura 8 de Ia presente invención. Todos los regímenes de tratamiento con ICOVIR-5 demostraron actividad oncolítica que resulta en una supresión del crecimiento tumoral significativamente distinta al grupo control (PBS), p<0,05. La administración de una pre-dosis de 3.1010 vp antes de Ia inyección de 1.1011 vp confiere a este régimen efectividad significativamente mejor que las otras pautas (p<0,05). Las diferentes secciones de los tumores congelados en OCT se trataron con una anticuerpo α-hexon (proteína de Ia cápsida del adenovirus) y se contratiñeron con 4',6'-diamin¡dino~2-phenyl¡ndol. La actividad antitumoral de ICOVIR-5 correlaciona con Ia replicación del adenovirus a nivel intratumoral, evaluada en los tumores obtenidos a día 22 post-inyección. Las muestras de todos los grupos tratados con ICOVI R-5 son positivas para Ia presencia de adenovirus, que localiza en áreas de necrosis tumoral.In another experiment the treatment was performed by systemic injection of ICOVIR5. Tumors of the SKMel-28 human melanoma cell line (1.10 7 cells / tumor) were implanted in BaIb C nu / nu nude mice, and once established they were treated by administration in the tail vein with PBS, with a single injection a day 0 of ICOVIR-5 of 2.5.10 10 viral particles (vp) or 1.10 11 vp, or with an injection of 3.10 10 vp and another one of 1.10 11 vp separated each other 1 hour. The results are shown in the lower part of Figure 8 of the present invention. All ICOVIR-5 treatment regimens demonstrated oncolytic activity resulting in a significantly different tumor growth suppression to the control group (PBS), p <0.05. The administration of a pre-dose of 3.10 10 vp before the injection of 1.10 11 vp gives this regimen significantly better effectiveness than the other guidelines (p <0.05). The different sections of the tumors frozen in OCT were treated with an α-hexon antibody (adenovirus capsid protein) and were counterstained with 4 ', 6'-diamininid ~ 2-phenylndol. The antitumor activity of ICOVIR-5 correlates with the replication of adenovirus at the intratumoral level, evaluated in tumors obtained at day 22 post-injection. The samples of all the groups treated with ICOVI R-5 are positive for the presence of adenovirus, which locates in areas of tumor necrosis.
EJEMPLO 6EXAMPLE 6
La toxicidad asociada a Ia administración sistémica de adenovirus se reduce cuando se utiliza un adenovirus que contiene E1a regulada con el promotor de E2F1 aislado con Ia secuencia DM y Ia secuencia de kozak en el inicio de traducción de E1a. La toxicidad in vivo de un adenovirus que contiene Ia secuencia de kozak en E1a y un promotor E2F1 aislado por DM (ICOVIR5) se comparó con Ia de un virus salvaje y el virus oncolítico AdD24RGD que expresa E1a bajo el promotor salvaje. Los virus se administraron endovenosamente a distintas dosis y a los 5 días post-inyección se evaluó parámetros asociados a Ia toxicidad como supervivencia animal, peso corporal, nivel de transaminasas séricas, y hematograma. Los resultados se muestran en Ia figura 9 de Ia presente invención. El valor de dosis letal 50 (LD50) para AdwtRGD ó AdΔ24RGD en ratones inmunocompetentes Balb/C se sitúa en 5.1010 partículas virales (vp)/ratón a día 5 post-inyección, mientras que el doble de esta dosis (1.1011 vp/ratón) sólo es letal para el 10% de los ratones (LD10) inyectados con ICOVIR-5. El peso corporal de los ratones inyectados con 5.1010 vp de AdwtRGD ó AdΔ24RGD a día 5 postinyección experimentó importantes pérdidas frente al incremento de peso de los ratones inyectados con ICOVIR-5. Paralelamente, las medidas de transaminasas hepáticas en plasma a día 5 post-inyección (valores medios ± S. D.; /i=5-10/ grupo) también revelaron importantes diferencias, siendo ICOVIR-5 claramente menos hepatotóxico a las mismas dosis. El perfil hematológico de los ratones a día 5 evidenció que Ia administración de 5.1010 vp de ICOVIR-5 no daba lugar a alteraciones significativas del hematograma ni reproducía Ia significativa plaquetopenia asociada a Ia administración de Ia misma dosis de AdwtRGD. El análisis de Ia expresión de Ia proteína adenoviral E1A en el hígado de los ratones por immunodetección en secciones congeladas obtenidas a día 5 post-inyección muestra que Ia presencia de una versión aislada del promotor de E2F-1 en ICOVIR-5 es efectiva restringiendo Ia expresión de las proteínas virales, incluso cuando Ia dosis administrada se incrementa (figura 10). La evaluación histológica por tinción de hematoxilina / eosina de secciones en parafina de los hígados a día 3 post-inyección también confirmó Ia baja toxicidad de ICOVIR-5 (figura 10). Así, mientras los hígados de los ratones que recibieron 5.1010 vp de AdwtRGD ó AdΔ24RGD presentaban síntomas claros de hepatitis fulminante (macroesteatosis, abundancia de cuerpos de Councilman, y presencia de puentes de necrosis), los animales inyectados con ICOVIR-5 tenían hígados con un fenotipo prácticamente normal, que sólo marginalmente presentaban cuerpos de Councilman en las regiones más externas.The toxicity associated with the systemic administration of adenovirus is reduced when an adenovirus containing E1a regulated with the E2F1 promoter isolated with the DM sequence and the kozak sequence is used at the beginning of E1a translation. The in vivo toxicity of an adenovirus containing the kozak sequence in E1a and an E2F1 promoter isolated by DM (ICOVIR5) was compared with that of a wild virus and the oncolytic virus AdD24RGD expressing E1a under the wild promoter. The viruses were administered intravenously at different doses and at 5 days post-injection parameters associated with toxicity such as animal survival, body weight, serum transaminase level, and hematogram were evaluated. The results are shown in Figure 9 of the present invention. The lethal dose value 50 (LD 50 ) for AdwtRGD or AdΔ24RGD in immunocompetent mice Balb / C stands at 5.10 10 viral particles (vp) / mouse at day 5 post-injection, while double this dose (1.10 11 vp / mouse) is only lethal for 10% of mice (LD 1 0) injected with ICOVIR-5. The body weight of the mice injected with 5.10 10 vp of AdwtRGD or AdΔ24RGD at day 5 post-injection experienced significant losses against the increase in weight of the mice injected with ICOVIR-5. At the same time, measures of liver transaminases in plasma at day 5 post-injection (mean values ± SD; / i = 5-10 / group) also revealed important differences, with ICOVIR-5 being clearly less hepatotoxic at the same doses. The hematological profile of the mice at day 5 showed that the administration of 5.10 10 vp of ICOVIR-5 did not give rise to significant alterations of the hematogram nor did it reproduce the significant platelet disease associated with the administration of the same dose of AdwtRGD. The analysis of the expression of adenoviral protein E1A in the liver of mice by immunodetection in frozen sections obtained on day 5 post-injection shows that the presence of an isolated version of the E2F-1 promoter in ICOVIR-5 is effective restricting Ia viral protein expression, even when the administered dose is increased (figure 10). Histological evaluation by hematoxylin / eosin staining of paraffin sections of the livers at day 3 post-injection also confirmed the low toxicity of ICOVIR-5 (Figure 10). Thus, while the livers of the mice that received 5.10 10 vp of AdwtRGD or AdΔ24RGD had clear symptoms of fulminant hepatitis (macro-steatosis, abundance of Councilman bodies, and presence of necrosis bridges), the animals injected with ICOVIR-5 had livers with a practically normal phenotype, which only marginally presented Councilman bodies in the outermost regions.
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Claims

REIVINDICACIONES
1. Adenovirus oncolítico para el tratamiento del cáncer caracterizado porque contiene una secuencia de DNA humano aislando a un promotor que confiere expresión selectiva a un gen adenoviral. 1. Oncolytic adenovirus for the treatment of cancer characterized in that it contains a human DNA sequence isolating a promoter that confers selective expression to an adenoviral gene.
2. Adenovirus oncolítico según Ia reivindicación 1 , caracterizado porque Ia secuencia de DNA humano es una secuencia derivada del locus de Ia distrofia miotónica.2. Oncolytic adenovirus according to claim 1, characterized in that the human DNA sequence is a sequence derived from the locus of myotonic dystrophy.
3. Adenovirus oncolítico según cualquiera de las reivindicaciones 1 a 2, caracterizado porque dicho adenovirus contiene una secuencia de kozak que optimiza Ia traducción proteica de un gen adenoviral regulado por un promotor que confiere selectividad tumoral.3. Oncolytic adenovirus according to any of claims 1 to 2, characterized in that said adenovirus contains a kozak sequence that optimizes the protein translation of an adenoviral gene regulated by a promoter that confers tumor selectivity.
4. Adenovirus oncolítico según cualquiera de las reivindicaciones 1 a 3, caracterizado porque contiene mutaciones en uno o más genes del grupo E1a, E1b y E4 para conseguir replicación selectiva en tumores. 4. Oncolytic adenovirus according to any of claims 1 to 3, characterized in that it contains mutations in one or more genes of the group E1a, E1b and E4 to achieve selective replication in tumors.
5. Adenovirus oncolítico según cualquiera de las reivindicaciones 1 a 3, caracterizado porque contiene modificaciones en su cápsida para aumentar su infectividad o dirigirlo a un receptor presente en una célula tumoral.5. Oncolytic adenovirus according to any of claims 1 to 3, characterized in that it contains modifications in its capsid to increase its infectivity or direct it to a receptor present in a tumor cell.
6. Adenovirus oncolítico según cualquiera de las reivindicaciones 1 a 3, caracterizado porque contiene otros genes usados comúnmente en el campo de terapia génica del cáncer como activadores de prodrogas, supresores tumorales o inmunoestimuladores.6. Oncolytic adenovirus according to any of claims 1 to 3, characterized in that it contains other genes commonly used in the field of cancer gene therapy such as prodrug activators, tumor suppressors or immunostimulators.
7. Adenovirus oncolítico según cualquiera de las reivindicaciones 1 a 3, caracterizado porque el adenovirus es un adenovirus humano derivado de un serotipo entre el 1 al 50. 7. Oncolytic adenovirus according to any of claims 1 to 3, characterized in that the adenovirus is a human adenovirus derived from a serotype between 1 and 50.
8. Adenovirus oncolítico según Ia reivindicación 7, caracterizado porque el adenovirus es un adenovirus humano del serotipo 5.8. Oncolytic adenovirus according to claim 7, characterized in that the adenovirus is a human adenovirus serotype 5.
9. Adenovirus oncolítico según cualquiera de las reivindicaciones 1 a 3, caracterizado porque el promotor que confiere selectividad tumoral es el promotor del gen E2F1 humano. 9. Oncolytic adenovirus according to any of claims 1 to 3, characterized in that the promoter that confers tumor selectivity is the promoter of the human E2F1 gene.
10. Adenovirus oncolítico según Ia reivindicación 9, caracterizado porque el promotor que confiere selectividad tumoral es un promotor E2F1 modificado por Ia inserción de sitios adicionales de unión a E2F.10. Oncolytic adenovirus according to claim 9, characterized in that the promoter that confers tumor selectivity is an E2F1 promoter modified by the insertion of additional E2F binding sites.
11. Composición farmacéutica que comprende una cantidad efectiva de un adenovirus oncolítico según cualquiera de las reivindicaciones 1 a 10 y uno o más portadores o excipientes farmacéuticamente aceptables.11. Pharmaceutical composition comprising an effective amount of an oncolytic adenovirus according to any of claims 1 to 10 and one or more pharmaceutically acceptable carriers or excipients.
12. Uso de un adenovirus oncolítico según cualquiera de las reivindicaciones 1 a 10 para Ia preparación de un medicamento para el tratamiento o prevención del cáncer o de una condición pre-maligna del mismo. 12. Use of an oncolytic adenovirus according to any of claims 1 to 10 for the preparation of a medicament for the treatment or prevention of cancer or a pre-malignant condition thereof.
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JP2010538652A (en) * 2007-09-13 2010-12-16 アボット・ラボラトリーズ Hepatitis B pre-S2 nucleic acid
WO2010097419A1 (en) 2009-02-25 2010-09-02 Fundació Privada Centre De Regulació Genòmica (Crg) Conditionally replicating adenovirus effective in the treatment of tumors
WO2010108931A1 (en) 2009-03-24 2010-09-30 Fundació Privada Institut D'investigació Biomèdica De Bellvitge (Idibell) Combination of an oncolytic adenovirus and a calcium channel blocker and its use for the treatment of cancer
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